@article {pmid38609262,
year = {2024},
author = {Su, Z and Wang, X and Chen, X and Ding, L and Zeng, X and Xu, J and Peng, C},
title = {Novel CRISPR/SpRY system for rapid detection of CRISPR/Cas-mediated gene editing in rice.},
journal = {Analytica chimica acta},
volume = {1303},
number = {},
pages = {342519},
doi = {10.1016/j.aca.2024.342519},
pmid = {38609262},
issn = {1873-4324},
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Biological Assay ; Biotechnology ; RNA ; },
abstract = {The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.},
}

*Oryza/genetics
CRISPR-Cas Systems/genetics
Gene Editing
Biological Assay
Biotechnology
RNA

@article {pmid38609257,
year = {2024},
author = {He, W and Li, X and Li, X and Guo, M and Zhang, M and Hu, R and Li, M and Ding, S and Yan, Y},
title = {Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA.},
journal = {Analytica chimica acta},
volume = {1303},
number = {},
pages = {342477},
doi = {10.1016/j.aca.2024.342477},
pmid = {38609257},
issn = {1873-4324},
mesh = {*MicroRNAs/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Nucleic Acids ; DNA, Single-Stranded/genetics ; },
abstract = {CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.},
}

*MicroRNAs/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems
*Nucleic Acids
DNA, Single-Stranded/genetics

@article {pmid38609223,
year = {2024},
author = {Ma, J and Qian, C and Hu, Q and Zhang, J and Gu, G and Liang, X and Zhang, L},
title = {The bacteriome-coupled phage communities continuously contract and shift to orchestrate the traditional rice vinegar fermentation.},
journal = {Food research international (Ottawa, Ont.)},
volume = {184},
number = {},
pages = {114244},
doi = {10.1016/j.foodres.2024.114244},
pmid = {38609223},
issn = {1873-7145},
mesh = {Humans ; *Oryza ; Acetic Acid ; Fermentation ; *Bacteriophages/genetics ; *Microbiota/genetics ; },
abstract = {Amounts of microbiome studies have uncovered the microbial communities of traditional food fermentations, while in which the phageome development with time is poorly understood. Here, we conducted a study to decipher both phageome and bacteriome of the traditional rice vinegar fermentation. The vinegar phageomes showed significant differences in the alpha diversity, network density and clustering coefficient over time. Peduoviridae had the highest relative abundance. Moreover, the phageome negatively correlated to the cognate bacteriome in alpha diversity, and undergone constantly contracting and shifting across the temporal scale. Nevertheless, 257 core virial clusters (VCs) persistently occurred with time whatever the significant impacts imposed by the varied physiochemical properties. Glycoside hydrolase (GH) and glycosyltransferase (GT) families genes displayed the higher abundances across all samples. Intriguingly, diversely structuring of toxin-antitoxin systems (TAs) and CRISPR-Cas arrays were frequently harbored by phage genomes. Their divergent organization and encoding attributes underlie the multiple biological roles in modulation of network and/or contest of phage community as well as bacterial host community. This phageome-wide mapping will fuel the current insights of phage community ecology in other traditional fermented ecosystems that are challenging to decipher.},
}

Humans
*Oryza
Acetic Acid
Fermentation
*Bacteriophages/genetics
*Microbiota/genetics

@article {pmid38607027,
year = {2024},
author = {Chua, R and Wang, L and Singaraja, R and Ghosh, S},
title = {Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.},
journal = {Cells},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/cells13070588},
pmid = {38607027},
issn = {2073-4409},
support = {2U54GM104940, R01HL146462-01/NH/NIH HHS/United States ; },
mesh = {Humans ; Animals ; Mice ; *Monocytes/metabolism ; *Furin/genetics/metabolism ; U937 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Multiomics ; RNA, Guide, CRISPR-Cas Systems ; Cytokines/genetics ; },
abstract = {The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.},
}

Humans
Animals
Mice
*Monocytes/metabolism
*Furin/genetics/metabolism
U937 Cells
Clustered Regularly Interspaced Short Palindromic Repeats
Multiomics
RNA, Guide, CRISPR-Cas Systems
Cytokines/genetics

@article {pmid38607007,
year = {2024},
author = {Wang, S and Zhu, Y and Du, S and Zheng, Y},
title = {Preclinical Advances in LNP-CRISPR Therapeutics for Solid Tumor Treatment.},
journal = {Cells},
volume = {13},
number = {7},
pages = {},
pmid = {38607007},
issn = {2073-4409},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; *Neoplasms/genetics/therapy ; *Nanostructures ; },
abstract = {Solid tumors, with their intricate cellular architecture and genetic heterogeneity, have long posed therapeutic challenges. The advent of the CRISPR genome editing system offers a promising, precise genetic intervention. However, the journey from bench to bedside is fraught with hurdles, chief among them being the efficient delivery of CRISPR components to tumor cells. Lipid nanoparticles (LNPs) have emerged as a potential solution. This biocompatible nanomaterial can encapsulate the CRISPR/Cas9 system, ensuring targeted delivery while mitigating off-target effects. Pre-clinical investigations underscore the efficacy of LNP-mediated CRISPR delivery, with marked disruption of oncogenic pathways and subsequent tumor regression. Overall, CRISPR/Cas9 technology, when combined with LNPs, presents a groundbreaking approach to cancer therapy, offering precision, efficacy, and potential solutions to current limitations. While further research and clinical testing are required, the future of personalized cancer treatment based on CRISPR/Cas9 holds immense promise.},
}

Humans
CRISPR-Cas Systems/genetics
Gene Editing
Genetic Therapy
*Neoplasms/genetics/therapy
*Nanostructures

@article {pmid38605715,
year = {2024},
author = {Shen, Q and Ruan, H and Zhang, H and Wu, T and Zhu, K and Han, W and Dong, R and Ming, T and Qi, H and Zhang, Y},
title = {Utilization of CRISPR-Cas genome editing technology in filamentous fungi: function and advancement potentiality.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1375120},
pmid = {38605715},
issn = {1664-302X},
abstract = {Filamentous fungi play a crucial role in environmental pollution control, protein secretion, and the production of active secondary metabolites. The evolution of gene editing technology has significantly improved the study of filamentous fungi, which in the past was laborious and time-consuming. But recently, CRISPR-Cas systems, which utilize small guide RNA (sgRNA) to mediate clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), have demonstrated considerable promise in research and application for filamentous fungi. The principle, function, and classification of CRISPR-Cas, along with its application strategies and research progress in filamentous fungi, will all be covered in the review. Additionally, we will go over general matters to take into account when editing a genome with the CRISPR-Cas system, including the creation of vectors, different transformation methodologies, multiple editing approaches, CRISPR-mediated transcriptional activation (CRISPRa) or interference (CRISPRi), base editors (BEs), and Prime editors (PEs).},
}

@article {pmid38578074,
year = {2024},
author = {Meng, JN and Xu, ZK and Li, PR and Zeng, X and Liu, Y and Xu, ZL and Wang, J and Ding, Y and Shen, X},
title = {Universal and Naked-Eye Diagnostic Platform for Emetic Bacillus cereus Based on RPA-Assisted CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {15},
pages = {8823-8830},
doi = {10.1021/acs.jafc.3c06744},
pmid = {38578074},
issn = {1520-5118},
mesh = {*Food Microbiology ; *Emetics ; Recombinases/genetics ; Bacillus cereus/genetics ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleotidyltransferases/genetics ; },
abstract = {Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10[-2] ng μL[-1] and 10[2] CFU mL[-1], respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.},
}

*Food Microbiology
*Emetics
Recombinases/genetics
Bacillus cereus/genetics
CRISPR-Cas Systems
Sensitivity and Specificity
Nucleotidyltransferases/genetics

@article {pmid38575365,
year = {2024},
author = {Wang, Y and Du, P and Shao, Y and Wang, W and Liu, Y and Ma, Y and Hu, P and Cao, J and Wang, X and Abd El-Aty, AM},
title = {An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {15},
pages = {8831-8839},
doi = {10.1021/acs.jafc.3c08829},
pmid = {38575365},
issn = {1520-5118},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; Coloring Agents ; DNA, Single-Stranded ; Recombinases ; Salmonella/genetics ; Polymerase Chain Reaction ; *Biosensing Techniques ; },
abstract = {Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 10[1]-2 × 10[9] CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.},
}

Animals
Cattle
*CRISPR-Cas Systems
Coloring Agents
DNA, Single-Stranded
Recombinases
Salmonella/genetics
Polymerase Chain Reaction
*Biosensing Techniques

@article {pmid38564120,
year = {2024},
author = {Sarkar, P and Santiago Vazquez, J and Zhou, M and Levy, A and Mou, Z and Orbović, V},
title = {Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.},
journal = {Transgenic research},
volume = {33},
number = {1-2},
pages = {59-66},
pmid = {38564120},
issn = {1573-9368},
support = {2018-70016-27392//USDA/NIFA Emergency Citrus Disease Research and Extension Program/ ; },
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Introns ; *Citrus/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Transfer/genetics ; },
abstract = {Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.},
}

*Gene Editing
CRISPR-Cas Systems/genetics
Introns
*Citrus/genetics
RNA, Guide, CRISPR-Cas Systems
RNA, Transfer/genetics

@article {pmid38504013,
year = {2024},
author = {Abkallo, HM and Arbuthnot, P and Auer, TO and Berger, DK and Burger, J and Chakauya, E and Concordet, JP and Diabate, A and Di Donato, V and Groenewald, JH and Guindo, A and Koekemoer, LL and Nazare, F and Nolan, T and Okumu, F and Orefuwa, E and Paemka, L and Prieto-Godino, L and Runo, S and Sadler, M and Tesfaye, K and Tripathi, L and Wondji, C},
title = {Making genome editing a success story in Africa.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {551-554},
pmid = {38504013},
issn = {1546-1696},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Africa ; Genome ; },
}

*Gene Editing
*CRISPR-Cas Systems/genetics
Africa
Genome

@article {pmid38497640,
year = {2024},
author = {Wang, P and Du, X and Zhao, Y and Wang, W and Cai, T and Tang, K and Wang, X},
title = {Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.},
journal = {Applied and environmental microbiology},
volume = {90},
number = {4},
pages = {e0009524},
pmid = {38497640},
issn = {1098-5336},
support = {32070175//MOST | National Natural Science Foundation of China (NSFC)/ ; 42188102//MOST | National Natural Science Foundation of China (NSFC)/ ; 91951203//MOST | National Natural Science Foundation of China (NSFC)/ ; 2022YFC3103600//MOST | National Key Research and Development Program of China (NKPs)/ ; 2019BT02Y262//Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program/ ; 2021345//Youth Innovation Promotion Association of the Chinese Academy of Sciences (CAS YIPA)/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Bacteria/genetics ; Genomic Islands ; Gene Transfer, Horizontal ; Plasmids/genetics ; Interspersed Repetitive Sequences ; },
abstract = {Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems
Bacteria/genetics
Genomic Islands
Gene Transfer, Horizontal
Plasmids/genetics
Interspersed Repetitive Sequences

@article {pmid38323580,
year = {2024},
author = {Romano, A and Mortellaro, A},
title = {The New Frontiers of Gene Therapy and Gene Editing in Inflammatory Diseases.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {219-231},
doi = {10.1089/hum.2023.210},
pmid = {38323580},
issn = {1557-7422},
mesh = {Animals ; Humans ; *Gene Editing/methods ; Quality of Life ; Genetic Therapy/methods ; Genetic Engineering ; *Severe Combined Immunodeficiency ; CRISPR-Cas Systems ; },
abstract = {Inflammatory diseases are conditions characterized by abnormal and often excessive immune responses, leading to tissue and organ inflammation. The complexity of these disorders arises from the intricate interplay of genetic factors and immune responses, which challenges conventional therapeutic approaches. However, the field of genetic manipulation has sparked unprecedented optimism in addressing these complex disorders. This review aims to comprehensively explore the application of gene therapy and gene editing in the context of inflammatory diseases, offering solutions that range from correcting genetic defects to precise immune modulation. These therapies have exhibited remarkable potential in ameliorating symptoms, improving quality of life, and even achieving disease remission. As we delve into recent breakthroughs and therapeutic applications, we illustrate how these advancements offer novel and transformative solutions for conditions that have traditionally eluded conventional treatments. By examining successful case studies and preclinical research, we emphasize the favorable results and substantial transformative impacts that gene-based interventions have demonstrated in patients and animal models of inflammatory diseases such as chronic granulomatous disease, cryopyrin-associated syndromes, and adenosine deaminase 2 deficiency, as well as those of multifactorial origins such as arthropathies (osteoarthritis, rheumatoid arthritis) and inflammatory bowel disease. In conclusion, gene therapy and gene editing offer transformative opportunities to address the underlying causes of inflammatory diseases, ushering in a new era of precision medicine and providing hope for personalized, targeted treatments.},
}

Animals
Humans
*Gene Editing/methods
Quality of Life
Genetic Therapy/methods
Genetic Engineering
*Severe Combined Immunodeficiency
CRISPR-Cas Systems

@article {pmid38251667,
year = {2024},
author = {Rai, R and Steinberg, Z and Romito, M and Zinghirino, F and Hu, YT and White, N and Naseem, A and Thrasher, AJ and Turchiano, G and Cavazza, A},
title = {CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {269-283},
doi = {10.1089/hum.2023.100},
pmid = {38251667},
issn = {1557-7422},
mesh = {Infant, Newborn ; Humans ; *Severe Combined Immunodeficiency/genetics/therapy ; T-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Hematopoietic Stem Cells/metabolism ; Gene Editing/methods ; Receptors, Interleukin-7/genetics/metabolism ; },
abstract = {Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets IL7RA exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.},
}

Infant, Newborn
Humans
*Severe Combined Immunodeficiency/genetics/therapy
T-Lymphocytes/metabolism
CRISPR-Cas Systems
Hematopoietic Stem Cells/metabolism
Gene Editing/methods
Receptors, Interleukin-7/genetics/metabolism

@article {pmid38159103,
year = {2024},
author = {Wang, R and Li, Y and Xu, S and Huang, Q and Tu, M and Zhu, Y and Cen, H and Dong, J and Jiang, L and Yao, X},
title = {Genome-wide association study reveals the genetic basis for petal-size formation in rapeseed (Brassica napus) and CRISPR-Cas9-mediated mutagenesis of BnFHY3 for petal-size reduction.},
journal = {The Plant journal : for cell and molecular biology},
volume = {118},
number = {2},
pages = {373-387},
doi = {10.1111/tpj.16609},
pmid = {38159103},
issn = {1365-313X},
support = {2022YFD1200404//National Key Research and Development Program of China/ ; },
mesh = {*Brassica napus/genetics ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Plant Breeding ; *Brassica rapa/genetics ; Mutagenesis ; },
abstract = {Petals in rapeseed (Brassica napus) serve multiple functions, including protection of reproductive organs, nutrient acquisition, and attraction of pollinators. However, they also cluster densely at the top, forming a thick layer that absorbs and reflects a considerable amount of photosynthetically active radiation. Breeding genotypes with large, small, or even petal-less varieties, requires knowledge of primary genes for allelic selection and manipulation. However, our current understanding of petal-size regulation is limited, and the lack of markers and pre-breeding materials hinders targeted petal-size breeding. Here, we conducted a genome-wide association study on petal size using 295 diverse accessions. We identified 20 significant single nucleotide polymorphisms and 236 genes associated with petal-size variation. Through a cross-analysis of genomic and transcriptomic data, we focused on 14 specific genes, from which molecular markers for diverging petal-size features can be developed. Leveraging CRISPR-Cas9 technology, we successfully generated a quadruple mutant of Far-Red Elongated Hypocotyl 3 (q-bnfhy3), which exhibited smaller petals compared to the wild type. Our study provides insights into the genetic basis of petal-size regulation in rapeseed and offers abundant potential molecular markers for breeding. The q-bnfhy3 mutant unveiled a novel role of FHY3 orthologues in regulating petal size in addition to previously reported functions.},
}

*Brassica napus/genetics
Genome-Wide Association Study
CRISPR-Cas Systems
Plant Breeding
*Brassica rapa/genetics
Mutagenesis

@article {pmid38069547,
year = {2024},
author = {Zhang, M and Feng, J and Li, Y and Qin, PZ and Chai, Y},
title = {Generation of tamoxifen-inducible Tfap2b-CreER[T2] mice using CRISPR-Cas9.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {62},
number = {1},
pages = {e23582},
pmid = {38069547},
issn = {1526-968X},
support = {R01 DE012711/NH/NIH HHS/United States ; R01 DE022503/NH/NIH HHS/United States ; R01 DE012711/DE/NIDCR NIH HHS/United States ; T90 DE021982/DE/NIDCR NIH HHS/United States ; R01 DE022503/DE/NIDCR NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Tamoxifen/pharmacology ; Mice, Transgenic ; Red Fluorescent Protein ; Integrases/genetics/metabolism ; },
abstract = {Tfap2b, a pivotal transcription factor, plays critical roles within neural crest cells and their derived lineage. To unravel the intricate lineage dynamics and contribution of these Tfap2b+ cells during craniofacial development, we established a Tfap2b-CreER[T2] knock-in transgenic mouse line using the CRISPR-Cas9-mediated homologous direct repair. By breeding with tdTomato reporter mice and initiating Cre activity through tamoxifen induction at distinct developmental time points, we show the Tfap2b lineage within the key neural crest-derived domains, such as the facial mesenchyme, midbrain, cerebellum, spinal cord, and limbs. Notably, the migratory neurons stemming from the dorsal root ganglia are visible subsequent to Cre activity initiated at E8.5. Intriguingly, Tfap2b+ cells, serving as the progenitors for limb development, show activity predominantly commencing at E10.5. Across the mouse craniofacial landscape, Tfap2b exhibits a widespread presence throughout the facial organs. Here we validate its role as a marker of progenitors in tooth development and have confirmed that this process initiates from E12.5. Our study not only validates the Tfap2b-CreER[T2] transgenic line, but also provides a powerful tool for lineage tracing and genetic targeting of Tfap2b-expressing cells and their progenitor in a temporally and spatially regulated manner during the intricate process of development and organogenesis.},
}

Mice
Animals
*CRISPR-Cas Systems
*Tamoxifen/pharmacology
Mice, Transgenic
Red Fluorescent Protein
Integrases/genetics/metabolism

@article {pmid38062734,
year = {2024},
author = {Whittaker, TE and Moula, SE and Bahal, S and Bakri, FG and Hayajneh, WA and Daoud, AK and Naseem, A and Cavazza, A and Thrasher, AJ and Santilli, G},
title = {Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67[phox]-Deficient Chronic Granulomatous Disease.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {298-312},
pmid = {38062734},
issn = {1557-7422},
support = {/WT_/Wellcome Trust/United Kingdom ; 104807/WT_/Wellcome Trust/United Kingdom ; MR/S021930/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Granulomatous Disease, Chronic/genetics/therapy ; Gene Editing ; Genetic Therapy/methods ; Antigens, CD34/genetics ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34[+] hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34[+] cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34[+] cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67[phox]-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67[phox] expression and functional correction of CD34[+]-derived neutrophils from a CGD patient.},
}

Humans
*Granulomatous Disease, Chronic/genetics/therapy
Gene Editing
Genetic Therapy/methods
Antigens, CD34/genetics
Hematopoietic Stem Cells/metabolism
CRISPR-Cas Systems

@article {pmid37997697,
year = {2024},
author = {Zhang, D and Boch, J},
title = {Development of TALE-adenine base editors in plants.},
journal = {Plant biotechnology journal},
volume = {22},
number = {5},
pages = {1067-1077},
pmid = {37997697},
issn = {1467-7652},
mesh = {Humans ; *Cytosine Deaminase/genetics ; *Adenine ; Gene Editing/methods ; DNA/genetics ; CRISPR-Cas Systems ; *Cytidine Deaminase ; *Proteins ; },
abstract = {Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes.},
}

Humans
*Cytosine Deaminase/genetics
*Adenine
Gene Editing/methods
DNA/genetics
CRISPR-Cas Systems
*Cytidine Deaminase
*Proteins

@article {pmid37400521,
year = {2024},
author = {Wessels, HH and Stirn, A and Méndez-Mancilla, A and Kim, EJ and Hart, SK and Knowles, DA and Sanjana, NE},
title = {Prediction of on-target and off-target activity of CRISPR-Cas13d guide RNAs using deep learning.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {628-637},
pmid = {37400521},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Deep Learning ; RNA ; Gene Editing ; },
abstract = {Transcriptome engineering applications in living cells with RNA-targeting CRISPR effectors depend on accurate prediction of on-target activity and off-target avoidance. Here we design and test ~200,000 RfxCas13d guide RNAs targeting essential genes in human cells with systematically designed mismatches and insertions and deletions (indels). We find that mismatches and indels have a position- and context-dependent impact on Cas13d activity, and mismatches that result in G-U wobble pairings are better tolerated than other single-base mismatches. Using this large-scale dataset, we train a convolutional neural network that we term targeted inhibition of gene expression via gRNA design (TIGER) to predict efficacy from guide sequence and context. TIGER outperforms the existing models at predicting on-target and off-target activity on our dataset and published datasets. We show that TIGER scoring combined with specific mismatches yields the first general framework to modulate transcript expression, enabling the use of RNA-targeting CRISPRs to precisely control gene dosage.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
*Deep Learning
RNA
Gene Editing

@article {pmid37365261,
year = {2024},
author = {},
title = {Efficient A-to-C base editing with high specificity.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {578-579},
pmid = {37365261},
issn = {1546-1696},
support = {32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; },
}

*Gene Editing
*CRISPR-Cas Systems/genetics

@article {pmid37322276,
year = {2024},
author = {Chen, L and Hong, M and Luan, C and Gao, H and Ru, G and Guo, X and Zhang, D and Zhang, S and Li, C and Wu, J and Randolph, PB and Sousa, AA and Qu, C and Zhu, Y and Guan, Y and Wang, L and Liu, M and Feng, B and Song, G and Liu, DR and Li, D},
title = {Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {638-650},
pmid = {37322276},
issn = {1546-1696},
support = {82100773//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; U01AI142756//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing ; *Adenine/metabolism ; Mutation ; Cytosine/metabolism ; Adenosine ; CRISPR-Cas Systems/genetics ; Mammals/genetics ; },
abstract = {Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.},
}

Animals
Mice
Humans
*Gene Editing
*Adenine/metabolism
Mutation
Cytosine/metabolism
Adenosine
CRISPR-Cas Systems/genetics
Mammals/genetics

@article {pmid38605260,
year = {2024},
author = {Rafiq, MS and Shabbir, MA and Raza, A and Irshad, S and Asghar, A and Maan, MK and Gondal, MA and Hao, H},
title = {CRISPR-Cas System: A New Dawn to Combat Antibiotic Resistance.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
pmid = {38605260},
issn = {1179-190X},
support = {32172914//the National Natural Science Foundation of China/ ; 2021YFD1800600//Key Technology Research and Development Program of Shandong Province/ ; . 2662022DKYJC005//the Fundamental Research Funds for the Central Universities/ ; },
abstract = {Antimicrobial resistance (AMR) can potentially harm global public health. Horizontal gene transfer (HGT), which speeds up the emergence of AMR and increases the burden of drug resistance in mobile genetic elements (MGEs), is the primary method by which AMR genes are transferred across bacterial pathogens. New approaches are urgently needed to halt the spread of bacterial diseases and antibiotic resistance. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an RNA-guided adaptive immune system, protects prokaryotes from foreign DNA like plasmids and phages. This approach may be essential in limiting horizontal gene transfer and halting the spread of antibiotic resistance. The CRISPR-Cas system has been crucial in identifying and understanding resistance mechanisms and developing novel therapeutic approaches. This review article investigates the CRISPR-Cas system's potential as a tool to combat bacterial AMR. Antibiotic-resistant bacteria can be targeted and eliminated by the CRISPR-Cas system. It has been proven to be an efficient method for removing carbapenem-resistant plasmids and regaining antibiotic susceptibility. The CRISPR-Cas system has enormous potential as a weapon against bacterial AMR. It precisely targets and eliminates antibiotic-resistant bacteria, facilitates resistance mechanism identification, and offers new possibilities in diagnostics and therapeutics.},
}

@article {pmid38605095,
year = {2024},
author = {de Boer, EN and Vroom, V and Scheper, AJ and Johansson, LF and Bosscher, L and Rietema, N and Commandeur-Jan, SZ and Knoers, NVAM and Sikkema-Raddatz, B and van den Berg, E and van Diemen, CC},
title = {Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8508},
pmid = {38605095},
issn = {2045-2322},
mesh = {Humans ; Reproducibility of Results ; *CRISPR-Cas Systems ; Karyotyping ; *Leukemia ; Chromosome Mapping ; },
abstract = {Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5-10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels or when labels are not distinct enough. Here, we test the potential of Cas9-directed long-read sequencing (LRS) as an additional technique to resolve such potentially relevant new findings. From an internal Bionano implementation study we selected ten OGM calls that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION/GridION device. We could confirm all aberrations and, importantly, the actual breakpoints of the OGM calls were located between 0.2 and 5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. Our results suggest that Cas9-directed LRS can be a relevant and flexible secondary technique in diagnostic workflows including OGM.},
}

Humans
Reproducibility of Results
*CRISPR-Cas Systems
Karyotyping
*Leukemia
Chromosome Mapping

@article {pmid38603815,
year = {2024},
author = {Bai, H and He, LY and Gao, FZ and Yao, KS and Zhang, M and Qiao, LK and Chen, ZY and He, LX and Liu, YS and Zhao, JL and Ying, GG},
title = {Airborne antibiotic resistome and microbiome in pharmaceutical factories.},
journal = {Environment international},
volume = {186},
number = {},
pages = {108639},
doi = {10.1016/j.envint.2024.108639},
pmid = {38603815},
issn = {1873-6750},
abstract = {Antimicrobial resistance is considered to be one of the biggest public health problems, and airborne transmission is an important but under-appreciated pathway for the spread of antibiotic resistance genes (ARGs) in the environment. Previous research has shown pharmaceutical factories to be a major source of ARGs and antibiotic resistant bacteria (ARB) in the surrounding receiving water and soil environments. Pharmaceutical factories are hotspots of antibiotic resistance, but the atmospheric transmission and its environmental risk remain more concerns. Here, we conducted a metagenomic investigation into the airborne microbiome and resistome in three pharmaceutical factories in China. Soil (average: 38.45%) and wastewater (average: 28.53%) were major contributors of airborne resistome. ARGs (vanR/vanS, blaOXA, and CfxA) conferring resistance to critically important clinically used antibiotics were identified in the air samples. The wastewater treatment area had significantly higher relative abundances of ARGs (average: 0.64 copies/16S rRNA). Approximately 28.2% of the detected airborne ARGs were found to be associated with plasmids, and this increased to about 50% in the wastewater treatment area. We have compiled a list of high-risk airborne ARGs found in pharmaceutical factories. Moreover, A total of 1,043 viral operational taxonomic units were identified and linked to 47 family-group taxa. Different CRISPR-Cas immune systems have been identified in bacterial hosts in response to phage infection. Similarly, higher phage abundance (average: 2451.70 PPM) was found in the air of the wastewater treatment area. Our data provide insights into the antibiotic resistance gene profiles and microbiome (bacterial and non-bacterial) in pharmaceutical factories and reveal the potential role of horizontal transfer in the spread of airborne ARGs, with implications for human and animal health.},
}

@article {pmid38602884,
year = {2024},
author = {van Beljouw, SPB and Haagsma, AC and Kalogeropoulos, K and Pabst, M and Brouns, SJJ},
title = {Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30.},
journal = {ACS chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acschembio.3c00788},
pmid = {38602884},
issn = {1554-8937},
abstract = {The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.},
}

@article {pmid38600465,
year = {2024},
author = {Ci, Y and Zhang, Y and Zhang, X},
title = {Methylated lncRNAs suppress apoptosis of gastric cancer stem cells via the lncRNA-miRNA/protein axis.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {51},
pmid = {38600465},
issn = {1689-1392},
support = {DY135-B-04//China Ocean Mineral Resources Research and Development Association/ ; },
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems ; Carcinogenesis/genetics ; Apoptosis/genetics ; Neoplastic Stem Cells/metabolism ; Cell Proliferation/genetics ; Gene Expression Regulation, Neoplastic ; Methyltransferases/genetics ; },
abstract = {BACKGROUND: Long noncoding RNAs (lncRNAs) play essential roles in the tumorigenesis of gastric cancer. However, the influence of lncRNA methylation on gastric cancer stem cells (GCSCs) remains unclear.

METHODS: The N6-methyladenosine (m6A) levels of lncRNAs in gastric cancer stem cells were detected by methylated RNA immunoprecipitation sequencing (MeRIP-seq), and the results were validated by MeRIP-quantitative polymerase chain reaction (qPCR). Specific sites of m6A modification on lncRNAs were detected by single-base elongation- and ligation-based qPCR amplification (SELECT). By constructing and transfecting the plasmid expressing methyltransferase-like 3 (METTL3) fused with catalytically inactivated Cas13 (dCas13b) and guide RNA targeting specific methylation sites of lncRNAs, we obtained gastric cancer stem cells with site-specific methylation of lncRNAs. Reverse transcription (RT)-qPCR and Western blot were used for detecting the stemness of treated gastric cancer stem cells.

RESULTS: The site-specific methylation of PSMA3-AS1 and MIR22HG suppressed apoptosis and promoted stemness of GCSCs. LncRNA methylation enhanced the stability of PSMA3-AS1 and MIR22HG to suppress apoptosis of GCSCs via the PSMA3-AS1-miR-411-3p- or MIR22HG-miR-24-3p-SERTAD1 axis. Simultaneously, the methylated lncRNAs promoted the interaction between PSMA3-AS1 and the EEF1A1 protein or MIR22HG and the LRPPRC protein, stabilizing the proteins and leading to the suppression of apoptosis. The in vivo data revealed that the methylated PSMA3-AS1 and MIR22HG triggered tumorigenesis of GCSCs.

CONCLUSIONS: Our study revealed the requirement for site-specific methylation of lncRNAs in the tumorigenesis of GCSCs, contributing novel insights into cancer development.},
}

Humans
*MicroRNAs/genetics/metabolism
*RNA, Long Noncoding/genetics/metabolism
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems
Carcinogenesis/genetics
Apoptosis/genetics
Neoplastic Stem Cells/metabolism
Cell Proliferation/genetics
Gene Expression Regulation, Neoplastic
Methyltransferases/genetics

@article {pmid38600182,
year = {2024},
author = {Moncaut, N},
title = {Streamlining mouse genome editing by integrating AAV repair template delivery and CRISPR-Cas electroporation.},
journal = {Lab animal},
volume = {},
number = {},
pages = {},
pmid = {38600182},
issn = {1548-4475},
}

@article {pmid38599852,
year = {2024},
author = {Liu, S and Zhang, Y and Luo, Y and Liu, J},
title = {Traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine.},
journal = {Journal of diabetes},
volume = {16},
number = {4},
pages = {e13545},
pmid = {38599852},
issn = {1753-0407},
support = {20202ACBL206008//Natural Science Foundation of Jiangxi Provincial/ ; 81760150//National Natural Science Foundation of China/ ; 82160162//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Regenerative Medicine ; Pancreas ; *Diabetes Mellitus, Type 1 ; Hepatocytes ; },
abstract = {Although pancreas and islet cell transplantation are the only ways to prevent the late complications of insulin-dependent diabetes, a shortage of donors is a major obstacle to tissue and organ transplantation. Stem cell therapy is an effective treatment for diabetes and other pancreatic-related diseases, which can be achieved by inducing their differentiation into insulin-secreting cells. The liver is considered an ideal source of pancreatic cells due to its similar developmental origin and strong regenerative ability as the pancreas. This article reviews the traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine and evaluates their advantages and challenges. Gene reprogramming and chemical reprogramming technologies are traditional strategies with potential to improve the efficiency and specificity of cell reprogramming and promote the transformation of hepatocytes into islet cells. At the same time, organoid technology, as an emerging strategy, has received extensive attention. Biomaterials provide a three-dimensional culture microenvironment for cells, which helps improve cell survival and differentiation efficiency. In addition, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has brought new opportunities and challenges to the development of organoid technology.},
}

Humans
*CRISPR-Cas Systems
Regenerative Medicine
Pancreas
*Diabetes Mellitus, Type 1
Hepatocytes

@article {pmid38599684,
year = {2024},
author = {Bukhari, H and Nithianadam, V and Battaglia, RA and Cicalo, A and Sarkar, S and Comjean, A and Hu, Y and Leventhal, MJ and Dong, X and Feany, MB},
title = {Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.},
journal = {Genome research},
volume = {},
number = {},
pages = {},
doi = {10.1101/gr.278576.123},
pmid = {38599684},
issn = {1549-5469},
abstract = {Missense mutations in the gene encoding the microtubule-associated protein tau cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human tau in experimental model organisms, including Drosophila, have been described. These models replicate key features of the human disease, but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas mediated gene editing to model frontotemporal dementia caused by the tau P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for tau P251L display age-dependent neurodegeneration, metabolic defects, and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies we performed single-cell RNA sequencing on approximately 130,000 cells from brains of tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified. Gene expression was also altered in glial cells, suggestive of non-cell autonomous regulation. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell type-specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy, which faithfully recapitulates the genetic context and phenotypic features of the human disease and use the results of comprehensive single cell sequencing analysis to outline pathways of neurotoxicity and highlight the potential role of non-cell autonomous changes in glia.},
}

@article {pmid38598861,
year = {2024},
author = {Burbano, DA and Kiattisewee, C and Karanjia, AV and Cardiff, RAL and Faulkner, ID and Sugianto, W and Carothers, JM},
title = {CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.},
journal = {Annual review of chemical and biomolecular engineering},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-chembioeng-100522-114706},
pmid = {38598861},
issn = {1947-5446},
abstract = {In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.},
}

@article {pmid38521283,
year = {2024},
author = {Hwang, J and Ye, DY and Jung, GY and Jang, S},
title = {Mobile genetic element-based gene editing and genome engineering: Recent advances and applications.},
journal = {Biotechnology advances},
volume = {72},
number = {},
pages = {108343},
doi = {10.1016/j.biotechadv.2024.108343},
pmid = {38521283},
issn = {1873-1899},
mesh = {*Gene Editing ; *Genetic Engineering ; Mutagenesis ; Interspersed Repetitive Sequences ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome engineering has revolutionized several scientific fields, ranging from biochemistry and fundamental research to therapeutic uses and crop development. Diverse engineering toolkits have been developed and used to effectively modify the genome sequences of organisms. However, there is a lack of extensive reviews on genome engineering technologies based on mobile genetic elements (MGEs), which induce genetic diversity within host cells by changing their locations in the genome. This review provides a comprehensive update on the versatility of MGEs as powerful genome engineering tools that offers efficient solutions to challenges associated with genome engineering. MGEs, including DNA transposons, retrotransposons, retrons, and CRISPR-associated transposons, offer various advantages, such as a broad host range, genome-wide mutagenesis, efficient large-size DNA integration, multiplexing capabilities, and in situ single-stranded DNA generation. We focused on the components, mechanisms, and features of each MGE-based tool to highlight their cellular applications. Finally, we discussed the current challenges of MGE-based genome engineering and provided insights into the evolving landscape of this transformative technology. In conclusion, the combination of genome engineering with MGE demonstrates remarkable potential for addressing various challenges and advancing the field of genetic manipulation, and promises to revolutionize our ability to engineer and understand the genomes of diverse organisms.},
}

*Gene Editing
*Genetic Engineering
Mutagenesis
Interspersed Repetitive Sequences
CRISPR-Cas Systems/genetics

@article {pmid38490381,
year = {2024},
author = {Sun, Z and Wang, M and Wang, W and Li, D and Wang, J and Sui, G},
title = {Getah virus capsid protein undergoes co-condensation with viral genomic RNA to facilitate virion assembly.},
journal = {International journal of biological macromolecules},
volume = {265},
number = {Pt 1},
pages = {130847},
doi = {10.1016/j.ijbiomac.2024.130847},
pmid = {38490381},
issn = {1879-0003},
mesh = {Animals ; Humans ; *Alphavirus/genetics/metabolism ; Capsid Proteins/genetics/metabolism ; RNA, Viral/genetics ; RNA, Guide, CRISPR-Cas Systems ; Genomics ; Virion/genetics ; },
abstract = {Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1-4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses.},
}

Animals
Humans
*Alphavirus/genetics/metabolism
Capsid Proteins/genetics/metabolism
RNA, Viral/genetics
RNA, Guide, CRISPR-Cas Systems
Genomics
Virion/genetics

@article {pmid38235591,
year = {2024},
author = {Ghahremani, S and Kanwal, A and Pettinato, A and Ladha, F and Legere, N and Thakar, K and Zhu, Y and Tjong, H and Wilderman, A and Stump, WT and Greenberg, L and Greenberg, MJ and Cotney, J and Wei, CL and Hinson, JT},
title = {CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants.},
journal = {Circulation},
volume = {149},
number = {16},
pages = {1285-1297},
doi = {10.1161/CIRCULATIONAHA.123.063972},
pmid = {38235591},
issn = {1524-4539},
support = {R01 HL141086/HL/NHLBI NIH HHS/United States ; R01 HL165220/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Cardiomyopathy, Dilated/genetics/therapy/pathology ; Connectin/genetics ; Haploinsufficiency/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; Myocytes, Cardiac/metabolism ; },
abstract = {BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity.

METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy-associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation.

RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts.

CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.},
}

Humans
*Cardiomyopathy, Dilated/genetics/therapy/pathology
Connectin/genetics
Haploinsufficiency/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RNA, Guide, CRISPR-Cas Systems
Myocytes, Cardiac/metabolism

@article {pmid38598855,
year = {2024},
author = {Hsieh, SC and Peters, JE},
title = {Natural and Engineered Guide RNA-directed Transposition with CRISPR-Associated Tn7-like Transposons.},
journal = {Annual review of biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-biochem-030122-041908},
pmid = {38598855},
issn = {1545-4509},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.},
}

@article {pmid38597925,
year = {2024},
author = {Li, J and Yan, K and Wang, S and Wang, P and Jiao, J and Dong, Y},
title = {Alteration of the intestinal microbiota and serum metabolites in a mouse model of Pon1 gene ablation.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {38},
number = {7},
pages = {e23611},
doi = {10.1096/fj.202302344R},
pmid = {38597925},
issn = {1530-6860},
mesh = {Animals ; Mice ; *Gastrointestinal Microbiome/genetics ; RNA, Guide, CRISPR-Cas Systems ; Disease Models, Animal ; *Microbiota ; Aryldialkylphosphatase/genetics ; Mice, Knockout ; },
abstract = {Mutations in the Paraoxonase 1 (Pon1) gene underlie aging, cardiovascular disease, and impairments of the nervous and gastrointestinal systems and are linked to the intestinal microbiome. The potential role of Pon1 in modulating the intestinal microbiota and serum metabolites is poorly understood. The present study demonstrated that mice with genomic excision of Pon1 by a multiplexed guide RNA CRISPR/Cas9 approach exhibited disrupted gut microbiota, such as significantly depressed alpha-diversity and distinctly separated beta diversity, accompanied by varied profiles of circulating metabolites. Furthermore, genomic knock in of Pon1 exerted a distinct effect on the intestinal microbiome and serum metabolome, including dramatically enriched Aerococcus, linoleic acid and depleted Bacillus, indolelactic acid. Specifically, a strong correlation was established between bacterial alterations and metabolites in Pon1 knockout mice. In addition, we identified metabolites related to gut bacteria in response to Pon1 knock in. Thus, the deletion of Pon1 affects the gut microbiome and functionally modifies serum metabolism, which can lead to dysbiosis, metabolic dysfunction, and infection risk. Together, these findings put forth a role for Pon1 in microbial alterations that contribute to metabolism variations. The function of Pon1 in diseases might at least partially depend on the microbiome.},
}

Animals
Mice
*Gastrointestinal Microbiome/genetics
RNA, Guide, CRISPR-Cas Systems
Disease Models, Animal
*Microbiota
Aryldialkylphosphatase/genetics
Mice, Knockout

@article {pmid38597776,
year = {2024},
author = {Xie, H and Su, F and Niu, Q and Geng, L and Cao, X and Song, M and Dong, J and Zheng, Z and Guo, R and Zhang, Y and Deng, Y and Ji, Z and Pang, K and Zhu, JK and Zhu, J},
title = {Knockout of miR396 genes increases seed size and yield in soybean.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13660},
pmid = {38597776},
issn = {1744-7909},
support = {2021LZGC003//Seed-Industrialized Development Program in Shandong Province, China/ ; 2022AH010056//the Innovative research team of Anhui education/ ; 2021LZGC012//Key Research & Development Program of Shandong Province, China/ ; 32270367//National Natural Science Foundation of China/ ; HSPHDSRF-2023-11-007//the PhD Scientific Research and Innovation Foundation of Sanya Yazhou Bay Science and Technology City/ ; NK2022010301//National Key Research and Development Program of China/ ; },
abstract = {Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss-of-function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.},
}

@article {pmid38414349,
year = {2024},
author = {Zeng, J and Liang, X and Duan, L and Tan, F and Chen, L and Qu, J and Li, J and Li, K and Luo, D and Hu, Z},
title = {Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.},
journal = {Acta biochimica et biophysica Sinica},
volume = {56},
number = {4},
pages = {525-537},
doi = {10.3724/abbs.2023280},
pmid = {38414349},
issn = {1745-7270},
mesh = {Humans ; Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems ; Fusion Proteins, bcr-abl/genetics/metabolism ; Genes, abl ; Proto-Oncogene Proteins c-bcr/genetics/metabolism ; CRISPR-Cas Systems ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/metabolism/therapy ; Apoptosis/genetics ; Cell Proliferation/genetics ; Drug Resistance, Neoplasm/genetics ; },
abstract = {The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein (BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase (ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.},
}

Humans
Animals
Mice
*RNA, Guide, CRISPR-Cas Systems
Fusion Proteins, bcr-abl/genetics/metabolism
Genes, abl
Proto-Oncogene Proteins c-bcr/genetics/metabolism
CRISPR-Cas Systems
*Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/metabolism/therapy
Apoptosis/genetics
Cell Proliferation/genetics
Drug Resistance, Neoplasm/genetics

@article {pmid38365083,
year = {2024},
author = {Duan, X and Li, H and Tan, X and Liu, N and Wang, X and Zhang, W and Liu, Y and Ma, W and Wu, Y and Ma, L and Fan, Y},
title = {Polygonum cillinerve polysaccharide inhibits transmissible gastroenteritis virus by regulating microRNA-181.},
journal = {Veterinary journal (London, England : 1997)},
volume = {304},
number = {},
pages = {106083},
doi = {10.1016/j.tvjl.2024.106083},
pmid = {38365083},
issn = {1532-2971},
mesh = {Animals ; Swine ; *Transmissible gastroenteritis virus/genetics ; *Polygonum/genetics ; RNA, Guide, CRISPR-Cas Systems ; Signal Transduction ; *MicroRNAs/genetics ; },
abstract = {Transmissible gastroenteritis virus (TGEV) is an important pathogen capable of altering the expression profile of cellular miRNA. In this study, the potential of Polygonum cillinerve polysaccharide (PCP) to treat TGEV-infected piglets was evaluated through in vivo experiments. High-throughput sequencing technology was employed to identify 9 up-regulated and 17 down-regulated miRNAs during PCP-mediated inhibition of TGEV infection in PK15 cells. Additionally, miR-181 was found to be associated with target genes of key proteins in the apoptosis pathway. PK15 cells were treated with various concentrations of PCP following transfection with miR-181 mimic or inhibitor. Real-time PCR assessed the impact on TGEV replication, while electron microscopy (TEM) and Hoechst fluorescence staining evaluated cellular functionality. Western blot analysis was utilized to assess the expression of key signaling factors-cytochrome C (cyt C), caspase 9, and P53-in the apoptotic signaling pathway. The results showed that compared with the control group, 250 μg/mL PCP significantly inhibited TGEV gRNA replication and gene N expression (P < 0.01). Microscopic examination revealed uniform cell morphology and fewer floating cells in PCP-treated groups (250 and 125 μg/mL). TEM analysis showed no typical virus structure in the 250 μg/mL PCP group, and apoptosis staining indicated a significant reduction in apoptotic cells at this concentration. Furthermore, PCP may inhibit TGEV-induced apoptosis via the Caspase-dependent mitochondrial pathway following miR-181 transfection. These findings provide a theoretical basis for further exploration into the mechanism of PCP's anti-TGEV properties.},
}

Animals
Swine
*Transmissible gastroenteritis virus/genetics
*Polygonum/genetics
RNA, Guide, CRISPR-Cas Systems
Signal Transduction
*MicroRNAs/genetics

@article {pmid38284432,
year = {2024},
author = {Bagheri, N and Chamorro, A and Idili, A and Porchetta, A},
title = {PAM-Engineered Toehold Switches as Input-Responsive Activators of CRISPR-Cas12a for Sensing Applications.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {63},
number = {17},
pages = {e202319677},
doi = {10.1002/anie.202319677},
pmid = {38284432},
issn = {1521-3773},
support = {MFAG 2022 - ID. 27151 project//Associazione Italiana per la Ricerca sul Cancro/ ; 2022FPYZ2N//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; 101086341//H2020 Marie Skłodowska-Curie Actions/ ; MSCA_0000010//European Union NextGenerationEU/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; DNA/metabolism ; Nucleic Acid Conformation ; },
abstract = {The RNA-programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio-engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity-based strand displacement reactions in response to target binding. By utilizing the trans-cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one-pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM-engineered Toehold Switches serve as programmable tools capable of regulating Cas12a-based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications.},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems
Gene Editing/methods
DNA/metabolism
Nucleic Acid Conformation

@article {pmid38589794,
year = {2024},
author = {Wang, Y and Zhai, Y and Zhang, M and Song, C and Zhang, Y and Zhang, G},
title = {Escaping from CRISPR-Cas-mediated knockout: the facts, mechanisms, and applications.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {48},
pmid = {38589794},
issn = {1689-1392},
support = {31970666//the National Natural Science Foundation of China/ ; tsqn201812054//Taishan Scholar Foundation of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR-Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR-Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR-Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR-Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods

@article {pmid38587910,
year = {2024},
author = {Sakamoto, N and Watanabe, K and Awazu, A and Yamamoto, T},
title = {CRISPR-Cas9-Mediated Gene Knockout in a Non-Model Sea Urchin, Heliocidaris crassispina.},
journal = {Zoological science},
volume = {41},
number = {2},
pages = {159-166},
doi = {10.2108/zs230052},
pmid = {38587910},
issn = {0289-0003},
mesh = {Animals ; *Anthocidaris/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gene Knockout Techniques ; Sea Urchins/genetics ; Gene Editing/methods ; },
abstract = {Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.},
}

Animals
*Anthocidaris/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems
Gene Knockout Techniques
Sea Urchins/genetics
Gene Editing/methods

@article {pmid38585710,
year = {2024},
author = {Xiao, Y and Qin, T and He, S and Chen, Y and Li, H and He, Q and Wang, X and Yang, S},
title = {Systematic investigation of TetR-family transcriptional regulators and their roles on lignocellulosic inhibitor acetate tolerance in Zymomonas mobilis.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {12},
number = {},
pages = {1385519},
pmid = {38585710},
issn = {2296-4185},
abstract = {TetR-family transcriptional regulators are widely distributed among bacteria and involved in various cellular processes such as multidrug and inhibitor resistance. Zymomonas mobilis is a industrial bacterium for lignocellulosic ethanol production. Although TetR-family regulators and their associated RND-family efflux pumps in Z. mobilis have been identified to be differentially expressed under various inhibitors and stressful conditions, there are no systematic investigation yet. In this study, bioinformatic analyses indicated that there are three TetR-family transcriptional regulators (ZMO0281, ZMO0963, ZMO1547) and two RND-family efflux pumps (ZMO0282-0285, ZMO0964-0966) adjacent to corresponding TetR-family regulators of ZMO0281 and ZMO0963 in Z. mobilis. Genetics studies were then carried out with various mutants of TetR-family regulators constructed, and ZMO0281 was characterized to be related to acetate tolerance. Combining transcriptomics and dual-reporter gene system, this study demonstrated that three TetR-family regulators repressed their adjacent genes specifically. Moreover, TetR-family regulator ZMO0281 might also be involved in other cellular processes in the presence of acetate. In addition, the upregulation of RND-family efflux pumps due to ZMO0281 deletion might lead to an energy imbalance and decreased cell growth in Z. mobilis under acetate stress. The systematic investigation of all three TetR-family regulators and their roles on a major lignocellulosic inhibitor acetate tolerance in Z. mobilis thus not only unravels the molecular mechanisms of TetR-family regulators and their potential cross-talks on regulating RND-family efflux pumps and other genes in Z. mobilis, but also provides guidance on understanding the roles of multiple regulators of same family in Z. mobilis and other microorganisms for efficient lignocellulosic biochemical production.},
}

@article {pmid38585687,
year = {2024},
author = {Fischer, J and Fedotova, A and Jaki, L and Sallard, E and Erhardt, A and Fuchs, J and Ruzsics, Z},
title = {Combining CRISPR-Cas-mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries.},
journal = {Molecular therapy. Methods & clinical development},
volume = {32},
number = {2},
pages = {101241},
pmid = {38585687},
issn = {2329-0501},
abstract = {While recombinant adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR-Cas9 mediated in vivo terminal resolution aiding high-efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of bacterial artificial chromosome-based rAd libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half site-directed fragment replacement (HFR) allows the introduction of more than 10[6] unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of approximately 2.5 × 10[4] unique rAds per cm[2] of transfected cell culture.},
}

@article {pmid38584940,
year = {2024},
author = {Carhuaricra-Huaman, D and Gonzalez, IHL and Ramos, PL and da Silva, AM and Setubal, JC},
title = {Analysis of twelve genomes of the bacterium Kerstersia gyiorum from brown-throated sloths (Bradypus variegatus), the first from a non-human host.},
journal = {PeerJ},
volume = {12},
number = {},
pages = {e17206},
pmid = {38584940},
issn = {2167-8359},
mesh = {Animals ; *Sloths/genetics ; Phylogeny ; Brazil ; *Alcaligenaceae/genetics ; },
abstract = {Kerstersia gyiorum is a Gram-negative bacterium found in various animals, including humans, where it has been associated with various infections. Knowledge of the basic biology of K. gyiorum is essential to understand the evolutionary strategies of niche adaptation and how this organism contributes to infectious diseases; however, genomic data about K. gyiorum is very limited, especially from non-human hosts. In this work, we sequenced 12 K. gyiorum genomes isolated from healthy free-living brown-throated sloths (Bradypus variegatus) in the Parque Estadual das Fontes do Ipiranga (São Paulo, Brazil), and compared them with genomes from isolates of human origin, in order to gain insights into genomic diversity, phylogeny, and host specialization of this species. Phylogenetic analysis revealed that these K. gyiorum strains are structured according to host. Despite the fact that sloth isolates were sampled from a single geographic location, the intra-sloth K. gyiorum diversity was divided into three clusters, with differences of more than 1,000 single nucleotide polymorphisms between them, suggesting the circulation of various K. gyiorum lineages in sloths. Genes involved in mobilome and defense mechanisms against mobile genetic elements were the main source of gene content variation between isolates from different hosts. Sloth-specific K. gyiorum genome features include an IncN2 plasmid, a phage sequence, and a CRISPR-Cas system. The broad diversity of defense elements in K. gyiorum (14 systems) may prevent further mobile element flow and explain the low amount of mobile genetic elements in K. gyiorum genomes. Gene content variation may be important for the adaptation of K. gyiorum to different host niches. This study furthers our understanding of diversity, host adaptation, and evolution of K. gyiorum, by presenting and analyzing the first genomes of non-human isolates.},
}

Animals
*Sloths/genetics
Phylogeny
Brazil
*Alcaligenaceae/genetics

@article {pmid38583547,
year = {2024},
author = {Saunier, M and Fortier, LC and Soutourina, O},
title = {RNA-based regulation in bacteria-phage interactions.},
journal = {Anaerobe},
volume = {87},
number = {},
pages = {102851},
doi = {10.1016/j.anaerobe.2024.102851},
pmid = {38583547},
issn = {1095-8274},
abstract = {Interactions of bacteria with their viruses named bacteriophages or phages shape the bacterial genome evolution and contribute to the diversity of phages. RNAs have emerged as key components of several anti-phage defense systems in bacteria including CRISPR-Cas, toxin-antitoxin and abortive infection. Frequent association with mobile genetic elements and interplay between different anti-phage defense systems are largely discussed. Newly discovered defense systems such as retrons and CBASS include RNA components. RNAs also perform their well-recognized regulatory roles in crossroad of phage-bacteria regulatory networks. Both regulatory and defensive function can be sometimes attributed to the same RNA molecules including CRISPR RNAs. This review presents the recent advances on the role of RNAs in the bacteria-phage interactions with a particular focus on clostridial species including an important human pathogen, Clostridioides difficile.},
}

@article {pmid38582932,
year = {2024},
author = {Wang, Y and Peng, Y and Zi, G and Chen, J and Peng, B},
title = {Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8095},
pmid = {38582932},
issn = {2045-2322},
mesh = {Humans ; Female ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/genetics/metabolism ; *Breast Neoplasms/genetics ; Gene Editing/methods ; },
abstract = {Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.},
}

Humans
Female
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems
RNA, Messenger/genetics/metabolism
*Breast Neoplasms/genetics
Gene Editing/methods

@article {pmid38582885,
year = {2024},
author = {Chan, YT and Wu, J and Lu, Y and Li, Q and Feng, Z and Xu, L and Yuan, H and Xing, T and Zhang, C and Tan, HY and Feng, Y and Wang, N},
title = {Loss of lncRNA LINC01056 leads to sorafenib resistance in HCC.},
journal = {Molecular cancer},
volume = {23},
number = {1},
pages = {74},
pmid = {38582885},
issn = {1476-4598},
support = {104006600//University Research Committee of The University of Hong Kong/ ; 17119621//Research Grant Committee of Hong Kong/ ; 19201591//The Health and Medical Research Fund/ ; PRP/028/22FX//Innovation and Technology Fund/ ; },
mesh = {Humans ; Mice ; Animals ; Sorafenib/pharmacology/therapeutic use ; *Carcinoma, Hepatocellular/drug therapy/genetics/metabolism ; *RNA, Long Noncoding/genetics ; *Liver Neoplasms/drug therapy/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; PPAR alpha/genetics/metabolism/therapeutic use ; Proteomics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND AND AIMS: Sorafenib is a major nonsurgical option for patients with advanced hepatocellular carcinoma (HCC); however, its clinical efficacy is largely undermined by the acquisition of resistance. The aim of this study was to identify the key lncRNA involved in the regulation of the sorafenib response in HCC.

MATERIALS AND METHODS: A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA) synergistic activation mediator (SAM)-pooled lncRNA library was applied to screen for the key lncRNA regulated by sorafenib treatment. The role of the identified lncRNA in mediating the sorafenib response in HCC was examined in vitro and in vivo. The underlying mechanism was delineated by proteomic analysis. The clinical significance of the expression of the identified lncRNA was evaluated by multiplex immunostaining on a human HCC microtissue array.

RESULTS: CRISPR/Cas9 lncRNA library screening revealed that Linc01056 was among the most downregulated lncRNAs in sorafenib-resistant HCC cells. Knockdown of Linc01056 reduced the sensitivity of HCC cells to sorafenib, suppressing apoptosis in vitro and promoting tumour growth in mice in vivo. Proteomic analysis revealed that Linc01056 knockdown in sorafenib-treated HCC cells induced genes related to fatty acid oxidation (FAO) while repressing glycolysis-associated genes, leading to a metabolic switch favouring higher intracellular energy production. FAO inhibition in HCC cells with Linc01056 knockdown significantly restored sensitivity to sorafenib. Mechanistically, we determined that PPARα is the critical molecule governing the metabolic switch upon Linc01056 knockdown in HCC cells and indeed, PPARα inhibition restored the sorafenib response in HCC cells in vitro and HCC tumours in vivo. Clinically, Linc01056 expression predicted optimal overall and progression-free survival outcomes in HCC patients and predicted a better sorafenib response. Linc01056 expression indicated a low FAO level in HCC.

CONCLUSION: Our study identified Linc01056 as a critical epigenetic regulator and potential therapeutic target in the regulation of the sorafenib response in HCC.},
}

Humans
Mice
Animals
Sorafenib/pharmacology/therapeutic use
*Carcinoma, Hepatocellular/drug therapy/genetics/metabolism
*RNA, Long Noncoding/genetics
*Liver Neoplasms/drug therapy/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems
PPAR alpha/genetics/metabolism/therapeutic use
Proteomics
Cell Line, Tumor
Drug Resistance, Neoplasm/genetics
Gene Expression Regulation, Neoplastic

@article {pmid38565025,
year = {2024},
author = {Zhu, Y and Lin, Y and Gong, B and Zhang, Y and Su, G and Yu, Y},
title = {Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping.},
journal = {Biosensors & bioelectronics},
volume = {255},
number = {},
pages = {116255},
doi = {10.1016/j.bios.2024.116255},
pmid = {38565025},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Genotype ; Polymorphism, Single Nucleotide/genetics ; *Biosensing Techniques ; Apolipoproteins E ; Uracil-DNA Glycosidase ; },
abstract = {Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.},
}

Humans
*CRISPR-Cas Systems/genetics
Genotype
Polymorphism, Single Nucleotide/genetics
*Biosensing Techniques
Apolipoproteins E
Uracil-DNA Glycosidase

@article {pmid38552526,
year = {2024},
author = {Yang, Y and Sun, L and Zhao, J and Jiao, Y and Han, T and Zhou, X},
title = {Improving trans-cleavage activity of CRISPR-Cas13a using engineered crRNA with a uridinylate-rich 5'-overhang.},
journal = {Biosensors & bioelectronics},
volume = {255},
number = {},
pages = {116239},
doi = {10.1016/j.bios.2024.116239},
pmid = {38552526},
issn = {1873-4235},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Biosensing Techniques ; RNA ; Recombinases ; SARS-CoV-2 ; CRISPR-Cas Systems/genetics ; },
abstract = {The engieering of Cas13a crRNA to enhance its binding affinity with the Cas enzyme or target is a promising method of improving the collateral cleavage efficiency of CRISPR-Cas13a systems, thereby amplifying the sensitivity of nucleic acid detection. An examination of the top-performing engineered crRNA (24 nt 5'7U LbuCas13a crRNA, where the 5'-end was extended using 7-mer uridinylates) and optimized conditions revealed an increased rate of LbuCas13a-mediated collateral cleavage activity that was up to seven-fold higher than that of the original crRNA. Particularly, the 7-mer uridinylates extension to crRNA was determined to be spacer-independent for enhancing the LbuCas13a-mediacted collateral cleavage activity, and also benefited the LwaCas13a system. The improved trans-cleavage activity was explained by the interactions between crRNA and LbuCas13a at the molecular level, i.e. the 5'-overhangs were anchored in the cleft formed between the Helical-1 and HEPN2 domains with the consequence of more stable complex, and experimentally verified. Consequently, the improved CRISPR-Cas13a system detected the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with a sensitivity of 2.36 fM that was 160-times higher than that of the original system. Using isothermal amplification via reverse transcription-recombinase polymerase amplification (RT-RPA), the system was capable to detect SARS-CoV-2 with attomolar sensitivity and accurately identified the SARS-CoV-2 Omicron variant (20/21 agreement) in clinical samples within 40 min.},
}

*RNA, Guide, CRISPR-Cas Systems
*Biosensing Techniques
RNA
Recombinases
SARS-CoV-2
CRISPR-Cas Systems/genetics

@article {pmid38504114,
year = {2024},
author = {Yao, D and Tycko, J and Oh, JW and Bounds, LR and Gosai, SJ and Lataniotis, L and Mackay-Smith, A and Doughty, BR and Gabdank, I and Schmidt, H and Guerrero-Altamirano, T and Siklenka, K and Guo, K and White, AD and Youngworth, I and Andreeva, K and Ren, X and Barrera, A and Luo, Y and Yardımcı, GG and Tewhey, R and Kundaje, A and Greenleaf, WJ and Sabeti, PC and Leslie, C and Pritykin, Y and Moore, JE and Beer, MA and Gersbach, CA and Reddy, TE and Shen, Y and Engreitz, JM and Bassik, MC and Reilly, SK},
title = {Multicenter integrated analysis of noncoding CRISPRi screens.},
journal = {Nature methods},
volume = {21},
number = {4},
pages = {723-734},
pmid = {38504114},
issn = {1548-7105},
support = {UM1 HG012053/HG/NHGRI NIH HHS/United States ; R00 HG009917/HG/NHGRI NIH HHS/United States ; U01 HG012103/HG/NHGRI NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; R01 HG010741/HG/NHGRI NIH HHS/United States ; R00 HG010669/HG/NHGRI NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; U24 HG009397/HG/NHGRI NIH HHS/United States ; R01 MH125236/MH/NIMH NIH HHS/United States ; U01 HG009380/HG/NHGRI NIH HHS/United States ; U01 HG009395/HG/NHGRI NIH HHS/United States ; U01 HG009431/HG/NHGRI NIH HHS/United States ; K00 DK126120/DK/NIDDK NIH HHS/United States ; UM1 HG009435/HG/NHGRI NIH HHS/United States ; R01 HG012872/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; U24 HG009446/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; Genome ; K562 Cells ; },
abstract = {The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.},
}

Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RNA, Guide, CRISPR-Cas Systems
Genome
K562 Cells

@article {pmid38499809,
year = {2024},
author = {Manjunath, LE and Singh, A and Devi Kumar, S and Vasu, K and Kar, D and Sellamuthu, K and Eswarappa, SM},
title = {Transcript-specific induction of stop codon readthrough using a CRISPR-dCas13 system.},
journal = {EMBO reports},
volume = {25},
number = {4},
pages = {2118-2143},
pmid = {38499809},
issn = {1469-3178},
support = {BT/PR38405/GET/119/309/2020//Department of Biotechnology, Ministry of Science and Technology, India (DBT)/ ; SB/SJF/2020-21/18//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; STARS/APR2019/BS/328/FS//Ministry of Education, Government of India/ ; ID-2021-13048//Indian Council of Medical Research (ICMR)/ ; },
mesh = {Animals ; Codon, Terminator/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Codon, Nonsense/genetics ; RNA, Messenger/genetics/metabolism ; Protein Biosynthesis ; Mammals/genetics/metabolism ; },
abstract = {Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.},
}

Animals
Codon, Terminator/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*RNA, Guide, CRISPR-Cas Systems
Codon, Nonsense/genetics
RNA, Messenger/genetics/metabolism
Protein Biosynthesis
Mammals/genetics/metabolism

@article {pmid38490564,
year = {2024},
author = {Pennati, A and Jakobi, M and Zeng, F and Ciampa, L and Rothbächer, U},
title = {Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.},
journal = {Developmental biology},
volume = {510},
number = {},
pages = {31-39},
doi = {10.1016/j.ydbio.2024.03.003},
pmid = {38490564},
issn = {1095-564X},
mesh = {Animals ; *Ciona intestinalis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Ciona/genetics ; Electroporation ; Gene Editing/methods ; },
abstract = {CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.},
}

Animals
*Ciona intestinalis/genetics/metabolism
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems
*Ciona/genetics
Electroporation
Gene Editing/methods

@article {pmid38412306,
year = {2024},
author = {Capin, J and Harrison, A and Raele, RA and Yadav, SKN and Baiwir, D and Mazzucchelli, G and Quinton, L and Satchwell, TJ and Toye, AM and Schaffitzel, C and Berger, I and Aulicino, F},
title = {An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing.},
journal = {Nucleic acids research},
volume = {52},
number = {6},
pages = {3450-3468},
pmid = {38412306},
issn = {1362-4962},
support = {834631/ERC_/European Research Council/International ; MR/V010506/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Mammals/genetics ; Baculoviridae/genetics ; DNA/genetics ; },
abstract = {CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.},
}

Animals
Humans
*Gene Editing
*CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
Mammals/genetics
Baculoviridae/genetics
DNA/genetics

@article {pmid38409225,
year = {2024},
author = {Montero, JJ and Trozzo, R and Sugden, M and Öllinger, R and Belka, A and Zhigalova, E and Waetzig, P and Engleitner, T and Schmidt-Supprian, M and Saur, D and Rad, R},
title = {Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx.},
journal = {Nature methods},
volume = {21},
number = {4},
pages = {584-596},
pmid = {38409225},
issn = {1548-7105},
support = {DFG RA1629/2-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB1321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB1371//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; (70114314)//Deutsche Krebshilfe (German Cancer Aid)/ ; Consolidator grant PACA-MET (819642)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; MSCA-ITNETN (861196//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; Long-Term Fellowship (ALFT 655-2019).//European Molecular Biology Organization (EMBO)/ ; },
mesh = {Humans ; Gene Expression Profiling ; *RNA, Long Noncoding/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Transcriptome ; *Neoplasms/genetics/metabolism ; },
abstract = {Although long noncoding RNAs (lncRNAs) dominate the transcriptome, their functions are largely unexplored. The extensive overlap of lncRNAs with coding and regulatory sequences restricts their systematic interrogation by DNA-directed perturbation. Here we developed genome-scale lncRNA transcriptome screening using Cas13d/CasRx. We show that RNA targeting overcomes limitations inherent to other screening methods, thereby considerably expanding the explorable space of the lncRNAome. By evolving the screening system toward pan-cancer applicability, it supports molecular and phenotypic data integration to contextualize screening hits or infer lncRNA function. We thereby addressed challenges posed by the enormous transcriptome size and tissue specificity through a size-reduced multiplexed gRNA library termed Albarossa, targeting 24,171 lncRNA genes. Its rational design incorporates target prioritization based on expression, evolutionary conservation and tissue specificity, thereby reconciling high discovery power and pan-cancer representation with scalable experimental throughput. Applied across entities, the screening platform identified numerous context-specific and common essential lncRNAs. Our work sets the stage for systematic exploration of lncRNA biology in health and disease.},
}

Humans
Gene Expression Profiling
*RNA, Long Noncoding/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems
Transcriptome
*Neoplasms/genetics/metabolism

@article {pmid38379122,
year = {2024},
author = {Li, S and Mei, L and He, C and Cai, X and Wu, H and Wu, X and Liu, Y and Feng, Y and Song, J},
title = {Identification of a family with van der Hoeve's syndrome harboring a novel COL1A1 mutation and generation of patient-derived iPSC lines and CRISPR/Cas9-corrected isogenic iPSCs.},
journal = {Human cell},
volume = {37},
number = {3},
pages = {817-831},
pmid = {38379122},
issn = {1749-0774},
support = {82071065//National Natural Science Foundation of China/ ; 2014CB541702//Major State Basic Research Development Program of China/ ; 2020YFC2005204//Key Technologies Research and Development Program/ ; 2020SK2106//Key Research and Development Program of Hunan Province of China/ ; 2021zzts0345//Fundamental Research Funds for Central Universities of the Central South University/ ; CX20210355//Fundamental Research Funds for Central Universities of the Central South University/ ; 2021M693566//Postdoctoral Research Foundation of China/ ; 2021T140751//Postdoctoral Research Foundation of China/ ; 2020RC2013//Science and Technology Program of Hunan Province/ ; 2021JJ41017//Natural Science Foundation of Hunan Province/ ; 2021JJ31084//Natural Science Foundation of Hunan Province/ ; 202107010047//Health Commission of Hunan Province/ ; },
mesh = {Humans ; *Osteogenesis Imperfecta/genetics/therapy ; Collagen Type I/genetics ; *Induced Pluripotent Stem Cells ; Leukocytes, Mononuclear ; CRISPR-Cas Systems/genetics ; Collagen Type I, alpha 1 Chain ; Mutation ; },
abstract = {Van der Hoeve's syndrome, also known as osteogenesis imperfecta (OI), is a genetic connective tissue disorder characterized by fragile, fracture-prone bone and hearing loss. The disease is caused by a gene mutation in one of the two type I collagen genes COL1A1 or COL1A2. In this study, we identified a novel frameshift mutation of the COL1A1 gene (c.1607delG) in a family with OI using whole-exome sequencing, bioinformatics analysis and Sanger sequencing. This mutation may lead to the deletion of a portion of exon 23 and the generation of a premature stop codon in the COL1A1 gene. To further investigate the impact of this mutation, we established two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells of OI patients carrying a novel mutation in the COL1A1 gene. Osteoblasts (OB) derived from OI-iPSCs exhibited reduced production of type I collagen and diminished ability to differentiate into osteoblasts. Using a CRISPR-based homology-directed repair strategy, we corrected the OI disease-causing COL1A1 novel mutations in iPSCs generated from an affected individual. Our results demonstrated that the diminished expression of type I collagen and osteogenic potential were enhanced in OB induced from corrected OI-iPSCs compared to those from OI-iPSCs. Overall, our results provide new insights into the genetic basis of Van der Hoeve's syndrome and highlight the potential of iPSC technology for disease modeling and therapeutic development.},
}

Humans
*Osteogenesis Imperfecta/genetics/therapy
Collagen Type I/genetics
*Induced Pluripotent Stem Cells
Leukocytes, Mononuclear
CRISPR-Cas Systems/genetics
Collagen Type I, alpha 1 Chain
Mutation

@article {pmid38347223,
year = {2024},
author = {Krink, N and Nikel, PI and Beisel, CL},
title = {A Hitchhiker's guide to CRISPR editing tools in bacteria : CRISPR can help unlock the bacterial world, but technical and regulatory barriers persist.},
journal = {EMBO reports},
volume = {25},
number = {4},
pages = {1694-1699},
pmid = {38347223},
issn = {1469-3178},
support = {NNF20OC0065068//Novo Nordisk Fonden (NNF)/ ; NNF20CC0035580//Novo Nordisk Fonden (NNF)/ ; NNF18OC0034818//Novo Nordisk Fonden (NNF)/ ; NNF21OC0067996//Novo Nordisk Fonden (NNF)/ ; 814418//EC | Horizon 2020 Framework Programme (H2020)/ ; 865973//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Bacteria/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Join us on a journey through the complex and ever-expanding universe of CRISPR approaches for genome editing in bacteria. Discover what is available, current technical challenges, successful implementation of these tools and the regulatory framework around their use. [Image: see text]},
}

*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Bacteria/genetics
Gene Editing
CRISPR-Cas Systems

@article {pmid38293815,
year = {2024},
author = {Wang, H and Tan, HY and Lian, J and Zhou, K},
title = {Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries.},
journal = {Biotechnology and bioengineering},
volume = {121},
number = {5},
pages = {1543-1553},
doi = {10.1002/bit.28664},
pmid = {38293815},
issn = {1097-0290},
support = {LR20B060003//Natural Science Foundation of Zhejiang Province/ ; 22278361//Natural Science Foundation of China/ ; 226-2023-00015//Fundamental Research Funds for the Central Universities/ ; 226-2022-00214//Fundamental Research Funds for the Central Universities/ ; 226-2023-00085//Fundamental Research Funds for the Central Universities/ ; //National Research Foundation of Singapore/ ; //Ministry of Education of Singapore/ ; //China Scholarship Council (CSC)/ ; //National University of Singapore/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; Saccharomyces cerevisiae/genetics/metabolism ; Transcriptional Activation ; *Nanopore Sequencing ; Cloning, Molecular ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in-house nanopore sequencing workflow was established for assessing the current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase-mediated non-amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a-based gene activation in Saccharomyces cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing-based workflow demonstrated here should provide a cost-effective approach for assessing the quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.},
}

*RNA, Guide, CRISPR-Cas Systems
Saccharomyces cerevisiae/genetics/metabolism
Transcriptional Activation
*Nanopore Sequencing
Cloning, Molecular
CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid38199243,
year = {2024},
author = {Song, N and Fan, X and Guo, X and Tang, J and Li, H and Tao, R and Li, F and Li, J and Yang, D and Yao, C and Liu, P},
title = {A DNA/Upconversion Nanoparticle Complex Enables Controlled Co-Delivery of CRISPR-Cas9 and Photodynamic Agents for Synergistic Cancer Therapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {36},
number = {15},
pages = {e2309534},
doi = {10.1002/adma.202309534},
pmid = {38199243},
issn = {1521-4095},
support = {22225505//National Natural Science Foundation of China/ ; 22322407//National Natural Science Foundation of China/ ; 32371468//National Natural Science Foundation of China/ ; //Innovative Research Team of High-level Local Universities in Shanghai/ ; },
mesh = {Mice ; Animals ; *Photochemotherapy ; CRISPR-Cas Systems ; Reactive Oxygen Species/metabolism ; Antioxidants ; Hemin ; Hydrogen Peroxide ; RNA, Guide, CRISPR-Cas Systems ; *Nanoparticles/therapeutic use ; Photosensitizing Agents/pharmacology/therapeutic use ; Hypoxia ; Cell Line, Tumor ; *Neoplasms/drug therapy ; },
abstract = {Photodynamic therapy (PDT) depends on the light-irradiated exciting of photosensitizer (PS) to generate reactive oxygen species (ROS), which faces challenges and limitations in hypoxia and antioxidant response of cancer cells, and limited tissue-penetration of light. Herein, a multifunctional DNA/upconversion nanoparticles (UCNPs) complex is developed which enables controlled co-delivery of CRISPR-Cas9, hemin, and protoporphyrin (PP) for synergistic PDT. An ultralong single-stranded DNA (ssDNA) is prepared via rolling circle amplification (RCA), which contains recognition sequences of single guide RNA (sgRNA) for loading Cas9 ribonucleoprotein (RNP), G-quadruplex sequences for loading hemin and PP, and linker sequences for combining UCNP. Cas9 RNP cleaves the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), improving the sensitivity of cancer cells to ROS, and enhancing the synergistic PDT effect. The G-quadruplex/hemin DNAzyme mimicks horseradish peroxidase (HRP) to catalyze the endogenous H2O2 to O2, overcoming hypoxia condition in tumors. The introduced UCNP converts NIR irradiation with deep tissue penetration to light with shorter wavelength, exciting PP to transform the abundant O2 to [1]O2. The integration of gene editing and PDT allows substantial accumulation of [1]O2 in cancer cells for enhanced cell apoptosis, and this synergistic PDT has shown remarkable therapeutic efficacy in a breast cancer mouse model.},
}

Mice
Animals
*Photochemotherapy
CRISPR-Cas Systems
Reactive Oxygen Species/metabolism
Antioxidants
Hemin
Hydrogen Peroxide
RNA, Guide, CRISPR-Cas Systems
*Nanoparticles/therapeutic use
Photosensitizing Agents/pharmacology/therapeutic use
Hypoxia
Cell Line, Tumor
*Neoplasms/drug therapy

@article {pmid38164916,
year = {2024},
author = {Yaylacıoğlu Tuncay, F and Talim, B and Dinçer, PR},
title = {Mimicking TGFBI Hot-Spot Mutation Did Not Result in Any Deposit Formation in the Zebrafish Cornea.},
journal = {Current eye research},
volume = {49},
number = {5},
pages = {458-466},
doi = {10.1080/02713683.2023.2298904},
pmid = {38164916},
issn = {1460-2202},
mesh = {Mice ; Animals ; Humans ; Transforming Growth Factor beta/genetics/metabolism ; Zebrafish/genetics ; RNA, Guide, CRISPR-Cas Systems ; Cornea/metabolism ; *Corneal Dystrophies, Hereditary/genetics ; Mutation ; *Corneal Opacity/metabolism ; Extracellular Matrix Proteins/genetics/metabolism ; DNA Mutational Analysis ; Pedigree ; },
abstract = {PURPOSE: Mutations in transforming growth factor beta-induced (TGFBI) protein are associated with a group of corneal dystrophies (CDs), classified as TGFBI-associated CDs, characterized by deposits in the cornea. Mouse models were not proper in several aspects for modelling human disease. The goal of this study was to generate zebrafish mutants to investigate the corneal phenotype and to decide whether zebrafish could be a potential model for TGFBI-associated CDs.

METHODS: The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3 months and 1 year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits.

RESULTS: We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L + del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3 months and 1 year of age.

CONCLUSION: This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1 year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.},
}

Mice
Animals
Humans
Transforming Growth Factor beta/genetics/metabolism
Zebrafish/genetics
RNA, Guide, CRISPR-Cas Systems
Cornea/metabolism
*Corneal Dystrophies, Hereditary/genetics
Mutation
*Corneal Opacity/metabolism
Extracellular Matrix Proteins/genetics/metabolism
DNA Mutational Analysis
Pedigree

@article {pmid38582601,
year = {2024},
author = {Koo, J and Zhu, GH and Palli, SR},
title = {CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.},
journal = {Pesticide biochemistry and physiology},
volume = {200},
number = {},
pages = {105839},
doi = {10.1016/j.pestbp.2024.105839},
pmid = {38582601},
issn = {1095-9939},
mesh = {Animals ; RNA Interference ; Spodoptera/genetics/metabolism ; *CRISPR-Cas Systems ; *RNA, Double-Stranded/genetics/metabolism ; Insecta/genetics ; Larva/genetics/metabolism ; },
abstract = {Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.},
}

Animals
RNA Interference
Spodoptera/genetics/metabolism
*CRISPR-Cas Systems
*RNA, Double-Stranded/genetics/metabolism
Insecta/genetics
Larva/genetics/metabolism

@article {pmid38582582,
year = {2024},
author = {Li, JL and Li, SS and Luo, ZJ and Lu, J and Cai, XM and Luo, ZX and Bian, L and Xiu, CL and Fu, NX and Liu, NY and Li, ZQ},
title = {CRISPR/Cas9-mediated ebony knockout causes melanin pigmentation and prevents moth Eclosion in Ectropis grisescens.},
journal = {Pesticide biochemistry and physiology},
volume = {200},
number = {},
pages = {105810},
doi = {10.1016/j.pestbp.2024.105810},
pmid = {38582582},
issn = {1095-9939},
mesh = {Animals ; *Moths/genetics ; Melanins/genetics ; CRISPR-Cas Systems ; Larva/genetics ; Pigmentation/genetics ; },
abstract = {Ectropis grisescens (Lepidoptera: Geometridae) is a destructive tea pest in China. Mimesis, characterized by changing body color, is an important trait of E. grisescens larvae. Hence, identifying melanin pathway-related genes may contribute to developing new pest control strategies. In the present study, we cloned Egebony, a gene potentially involved in melanin pigmentation in E. grisescens, and subsequently conducted CRISPR/Cas9-mediated targeted mutagenesis of Egebony to analyze its role in pigmentation and development. At the larvae, prepupae, and pupae stages, Egebony-knockout individuals exhibited darker pigmentation than the wild-type. However, Egebony knockout did not impact the colors of sclerotized appendants, including ocelli, setae, and claws. While mutant pupae could successfully develop into moths, they were unable to emerge from the puparium. Notably, embryo hatchability and larval survival of mutants remained normal. Further investigation indicated that mutant pupae exhibited significantly stronger shearing force than the wild-type, with the pigmented layer of mutant pupae appearing darker and thicker. Collectively, these results suggest that the loss of Egebony might increase the rigidity of the puparium and prevent moth eclosion. This study provides new insights into understanding the function and diversification of ebony in insect development and identifies a lethal gene that can be manipulated for developing effective pest control strategies.},
}

Animals
*Moths/genetics
Melanins/genetics
CRISPR-Cas Systems
Larva/genetics
Pigmentation/genetics

@article {pmid38581602,
year = {2024},
author = {Zhang, H and Liang, M and Chen, J and Wang, H and Ma, L},
title = {Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.},
journal = {Planta},
volume = {259},
number = {5},
pages = {112},
pmid = {38581602},
issn = {1432-2048},
support = {LY24C130004//Natural Science Foundation of Zhejiang Province/ ; LY22C135104//Natural Science Foundation of Zhejiang Province/ ; LY23C130002//Natural Science Foundation of Zhejiang Province/ ; 31501288//National Natural Science Foundation of China/ ; 20210207//Open Project Program of State Key Laboratory of Rice Biology/ ; CPSIBRF-CNRRI-202306//Central Public-interest Scientific Institution Basal Research Fund/ ; },
mesh = {*CRISPR-Cas Systems ; *Oryza/genetics ; Disease Resistance/genetics ; Odorants ; Temperature ; Plant Breeding ; },
abstract = {The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.},
}

*CRISPR-Cas Systems
*Oryza/genetics
Disease Resistance/genetics
Odorants
Temperature
Plant Breeding

@article {pmid38580329,
year = {2024},
author = {Lo Presti, V and Meringa, A and Dunnebach, E and van Velzen, A and Moreira, AV and Stam, RW and Kotecha, RS and Krippner-Heidenreich, A and Heidenreich, OT and Plantinga, M and Cornel, A and Sebestyen, Z and Kuball, J and van Til, NP and Nierkens, S},
title = {Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML.},
journal = {Journal for immunotherapy of cancer},
volume = {12},
number = {4},
pages = {},
pmid = {38580329},
issn = {2051-1426},
mesh = {Humans ; Child ; CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems/genetics ; Fetal Blood ; Receptors, Antigen, T-Cell/genetics ; *Leukemia, Myeloid, Acute/genetics/therapy ; Cell Line, Tumor ; *Antineoplastic Agents ; Recurrence ; },
abstract = {BACKGROUND: Hematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due to relapse. To improve overall survival, we propose a combined strategy based on cord blood (CB)-HCT with the application of AML-specific T cell receptor (TCR)-engineered T cell therapy derived from the same CB graft.

METHODS: We produced CB-CD8[+] T cells expressing a recombinant TCR (rTCR) against Wilms tumor 1 (WT1) while lacking endogenous TCR (eTCR) expression to avoid mispairing and competition. CRISPR-Cas9 multiplexing was used to target the constant region of the endogenous TCRα (TRAC) and TCRβ (TRBC) chains. Next, an optimized method for lentiviral transduction was used to introduce recombinant WT1-TCR. The cytotoxic and migration capacity of the product was evaluated in coculture assays for both cell lines and primary pediatric AML blasts.

RESULTS: The gene editing and transduction procedures achieved high efficiency, with up to 95% of cells lacking eTCR and over 70% of T cells expressing rWT1-TCR. WT1-TCR-engineered T cells lacking the expression of their eTCR (eTCR[-/-] WT1-TCR) showed increased cell surface expression of the rTCR and production of cytotoxic cytokines, such as granzyme A and B, perforin, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), on antigen recognition when compared with WT1-TCR-engineered T cells still expressing their eTCR (eTCR[+/+] WT1-TCR). CRISPR-Cas9 editing did not affect immunophenotypic characteristics or T cell activation and did not induce increased expression of inhibitory molecules. eTCR[-/-] WT1-TCR CD8[+] CB-T cells showed effective migratory and killing capacity in cocultures with neoplastic cell lines and primary AML blasts, but did not show toxicity toward healthy cells.

CONCLUSIONS: In summary, we show the feasibility of developing a potent CB-derived CD8[+] T cell product targeting WT1, providing an option for post-transplant allogeneic immune cell therapy or as an off-the-shelf product, to prevent relapse and improve the clinical outcome of children with AML.},
}

Humans
Child
CD8-Positive T-Lymphocytes
CRISPR-Cas Systems/genetics
Fetal Blood
Receptors, Antigen, T-Cell/genetics
*Leukemia, Myeloid, Acute/genetics/therapy
Cell Line, Tumor
*Antineoplastic Agents
Recurrence

@article {pmid38579680,
year = {2024},
author = {Cavazzana, M and Miccio, A},
title = {The difficult translational pathway from animal models to patients.},
journal = {Cell stem cell},
volume = {31},
number = {4},
pages = {435-436},
doi = {10.1016/j.stem.2024.03.010},
pmid = {38579680},
issn = {1875-9777},
mesh = {Animals ; Humans ; *Gene Editing ; Hematopoietic Stem Cells/metabolism ; Models, Animal ; CRISPR-Cas Systems/genetics ; *Hematopoietic Stem Cell Transplantation ; },
abstract = {Lee et al.[1] analyzed the impacts of lentiviral vector transduction and CRISPR-Cas9/homology-directed repair editing on hematopoietic stem and progenitor cell (HSPC) engraftment and clonal dynamics. The study suggests that relative to lentiviral-vector-mediated gene addition, homology-directed repair editing is inefficient in vivo and might impair the engraftment and differentiation of HSPCs.},
}

Animals
Humans
*Gene Editing
Hematopoietic Stem Cells/metabolism
Models, Animal
CRISPR-Cas Systems/genetics
*Hematopoietic Stem Cell Transplantation

@article {pmid38579669,
year = {2024},
author = {Jalil, S and Keskinen, T and Juutila, J and Sartori Maldonado, R and Euro, L and Suomalainen, A and Lapatto, R and Kuuluvainen, E and Hietakangas, V and Otonkoski, T and Hyvönen, ME and Wartiovaara, K},
title = {Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors.},
journal = {American journal of human genetics},
volume = {111},
number = {4},
pages = {714-728},
doi = {10.1016/j.ajhg.2024.03.004},
pmid = {38579669},
issn = {1537-6605},
mesh = {Humans ; *Argininosuccinic Aciduria/genetics ; Argininosuccinate Lyase/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems ; Urea ; },
abstract = {Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.},
}

Humans
*Argininosuccinic Aciduria/genetics
Argininosuccinate Lyase/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide, CRISPR-Cas Systems
Urea

@article {pmid38576085,
year = {2024},
author = {Song, J and Luo, N and Dong, L and Peng, J and Yi, C},
title = {RNA base editors: The emerging approach of RNA therapeutics.},
journal = {Wiley interdisciplinary reviews. RNA},
volume = {15},
number = {2},
pages = {e1844},
doi = {10.1002/wrna.1844},
pmid = {38576085},
issn = {1757-7012},
support = {2023YFC3402200//Ministry of Science and Technology of the People's Republic of China/ ; 2019YFA0802200//Ministry of Science and Technology of the People's Republic of China/ ; 92153303//National Natural Science Foundation of China/ ; 21825701//National Natural Science Foundation of China/ ; Z231100002723005//Beijing Municipal Science and Technology Commission/ ; },
mesh = {*RNA/genetics ; *CRISPR-Cas Systems ; Gene Editing ; Oligonucleotides, Antisense ; RNA Interference ; },
abstract = {RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m[6]A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development.},
}

*RNA/genetics
*CRISPR-Cas Systems
Gene Editing
Oligonucleotides, Antisense
RNA Interference

@article {pmid38575718,
year = {2024},
author = {Ren, K and Zhou, F and Zhang, F and Yin, M and Zhu, Y and Wang, S and Chen, Y and Huang, T and Wu, Z and He, J and Zhang, A and Guo, C and Huang, Z},
title = {Discovery and structural mechanism of DNA endonucleases guided by RAGATH-18-derived RNAs.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
pmid = {38575718},
issn = {1748-7838},
support = {31825008//National Natural Science Foundation of China (National Science Foundation of China)/ ; U21A20276//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteria IS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.},
}

@article {pmid37461250,
year = {2024},
author = {Chen, F and Guo, H and Lan, W and Zhou, M and Geng, W and Shen, G and Lin, P and Xia, Q and Zhao, P and Li, Z},
title = {Targeted DNA N[6]-methyladenine editing by dCas9 fused to METTL4 in the lepidopteran model insect Bombyx mori.},
journal = {Insect science},
volume = {31},
number = {2},
pages = {646-650},
doi = {10.1111/1744-7917.13222},
pmid = {37461250},
issn = {1744-7917},
support = {cstc2020jcyj-cxttX0001//Natural Science Foundation of Chongqing/ ; 2022YFD1201600//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Bombyx/genetics ; DNA Methylation ; DNA/genetics ; CRISPR-Cas Systems ; *Adenine ; },
abstract = {We have established a novel CRISPR-dCas9-METTL4 epigenome editing tool that can methylate target regions to achieve site-specific DNA 6mA methylation in both hypermethylated and hypomethylated genes. Targeted methylation on genes by dCas9-METTL4 results in misexpression, allowing for the functional investigation of target genes of interest in silkworm.},
}

Animals
*Bombyx/genetics
DNA Methylation
DNA/genetics
CRISPR-Cas Systems
*Adenine

@article {pmid38575625,
year = {2024},
author = {Flores, VS and Amgarten, DE and Iha, BKV and Ryon, KA and Danko, D and Tierney, BT and Mason, C and da Silva, AM and Setubal, JC},
title = {Discovery and description of novel phage genomes from urban microbiomes sampled by the MetaSUB consortium.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {7913},
pmid = {38575625},
issn = {2045-2322},
mesh = {*Bacteriophages/genetics ; *Railroads ; *Microbiota/genetics ; Metagenome/genetics ; Bacteria/genetics ; },
abstract = {Bacteriophages are recognized as the most abundant members of microbiomes and have therefore a profound impact on microbial communities through the interactions with their bacterial hosts. The International Metagenomics and Metadesign of Subways and Urban Biomes Consortium (MetaSUB) has sampled mass-transit systems in 60 cities over 3 years using metagenomics, throwing light into these hitherto largely unexplored urban environments. MetaSUB focused primarily on the bacterial community. In this work, we explored MetaSUB metagenomic data in order to recover and analyze bacteriophage genomes. We recovered and analyzed 1714 phage genomes with size at least 40 kbp, from the class Caudoviricetes, the vast majority of which (80%) are novel. The recovered genomes were predicted to belong to temperate (69%) and lytic (31%) phages. Thirty-three of these genomes have more than 200 kbp, and one of them reaches 572 kbp, placing it among the largest phage genomes ever found. In general, the phages tended to be site-specific or nearly so, but 194 genomes could be identified in every city from which phage genomes were retrieved. We predicted hosts for 48% of the phages and observed general agreement between phage abundance and the respective bacterial host abundance, which include the most common nosocomial multidrug-resistant pathogens. A small fraction of the phage genomes are carriers of antibiotic resistance genes, and such genomes tended to be particularly abundant in the sites where they were found. We also detected CRISPR-Cas systems in five phage genomes. This study expands the previously reported MetaSUB results and is a contribution to the knowledge about phage diversity, global distribution, and phage genome content.},
}

*Bacteriophages/genetics
*Railroads
*Microbiota/genetics
Metagenome/genetics
Bacteria/genetics

@article {pmid38575571,
year = {2024},
author = {Zhao, H and Sheng, Y and Zhang, T and Zhou, S and Zhu, Y and Qian, F and Liu, M and Xu, W and Zhang, D and Hu, J},
title = {The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {2901},
pmid = {38575571},
issn = {2041-1723},
support = {2022A1515010370//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; 2022A1515011721//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; },
mesh = {Animals ; Humans ; RNA/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; *Epstein-Barr Virus Infections/genetics ; Herpesvirus 4, Human/genetics ; *MicroRNAs ; Mammals/genetics ; },
abstract = {Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.},
}

Animals
Humans
RNA/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems/genetics
*Epstein-Barr Virus Infections/genetics
Herpesvirus 4, Human/genetics
*MicroRNAs
Mammals/genetics

@article {pmid38573978,
year = {2024},
author = {Zheng, C and Liang, H and Dai, L and Yu, J and Long, C},
title = {Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.},
journal = {The journal of physical chemistry. B},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jpcb.3c07320},
pmid = {38573978},
issn = {1520-5207},
abstract = {Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.},
}

@article {pmid38573530,
year = {2024},
author = {Zhao, Z and Cao, S and Sun, M and Yang, Q and Huang, T and Yang, X and Li, J and Zhang, X and Li, X and Wang, X and Jiang, W and Gong, P},
title = {Rapid visual detection of Giardia duodenalis in faecal samples using an RPA-CRISPR/Cas12a system.},
journal = {Parasitology research},
volume = {123},
number = {4},
pages = {176},
pmid = {38573530},
issn = {1432-1955},
support = {20220508101RC//Jilin Scientific and Technological Development Program/ ; 21ZY24//Changchun Science and Technology Program/ ; CARS-39//China Wool-sheep & Cashmere-goat Research System/ ; },
mesh = {Humans ; Animals ; Cattle ; *Giardia lamblia/genetics ; CRISPR-Cas Systems ; *Giardiasis/diagnosis/veterinary ; Giardia/genetics ; Biological Assay ; },
abstract = {Giardiasis is a common intestinal infection caused by Giardia duodenalis, which is a major economic and health burden for humans and livestock. Currently, a convenient and effective detection method is urgently needed. CRISPR/Cas12a-based diagnostic methods have been widely used for nucleic acid-based detection of pathogens due to their high efficiency and sensitivity. In this study, a technique combining CRISPR/Cas12a and RPA was established that allows the detection of G. duodenalis in faecal samples by the naked eye with high sensitivity (10[-1] copies/μL) and specificity (no cross-reactivity with nine common pathogens). In clinical evaluations, the RPA-CRISPR/Cas12a-based detection assay detected Giardia positivity in 2% (1/50) of human faecal samples and 47% (33/70) of cattle faecal samples, respectively, which was consistent with the results of nested PCR. Our study demonstrated that the RPA-CRISPR/Cas12a technique for G. duodenalis is stable, efficient, sensitive, specific and has low equipment requirements. This technique offers new opportunities for on-site detection in remote and poor areas.},
}

Humans
Animals
Cattle
*Giardia lamblia/genetics
CRISPR-Cas Systems
*Giardiasis/diagnosis/veterinary
Giardia/genetics
Biological Assay

@article {pmid38573428,
year = {2024},
author = {Calbry, J and Goudounet, G and Charlot, F and Guyon-Debast, A and Perroud, PF and Nogué, F},
title = {The SpRY Cas9 variant release the PAM sequence constraint for genome editing in the model plant Physcomitrium patens.},
journal = {Transgenic research},
volume = {},
number = {},
pages = {},
pmid = {38573428},
issn = {1573-9368},
support = {ANR-20-CE20-0027-02-TOR-DYRKcontrol//Agence Nationale de la Recherche/ ; ANR-17-EUR-0007//Agence Nationale de la Recherche/ ; ANR-17-EUR-0007//Agence Nationale de la Recherche/ ; ANR-20-CE20-0027-02-TOR-DYRKcontrol//Agence Nationale de la Recherche/ ; },
abstract = {Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants.},
}

@article {pmid38556749,
year = {2024},
author = {You, S and Yao, S and Chen, X and Hou, Q and Liu, Z and Lei, G and Xie, X and Liang, Z and Yuchi, Z and You, M and Liu, Y and Xiong, L},
title = {CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {14},
pages = {8180-8188},
doi = {10.1021/acs.jafc.3c08721},
pmid = {38556749},
issn = {1520-5118},
mesh = {Animals ; Female ; *Moths/genetics/metabolism ; Larva/metabolism ; *Bacillus thuringiensis/genetics/metabolism ; Longevity ; CRISPR-Cas Systems ; Endotoxins/genetics/metabolism ; Bacillus thuringiensis Toxins/metabolism ; Bacterial Proteins/genetics/metabolism ; Hemolysin Proteins/genetics/metabolism ; Insecticide Resistance/genetics ; },
abstract = {Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.},
}

Animals
Female
*Moths/genetics/metabolism
Larva/metabolism
*Bacillus thuringiensis/genetics/metabolism
Longevity
CRISPR-Cas Systems
Endotoxins/genetics/metabolism
Bacillus thuringiensis Toxins/metabolism
Bacterial Proteins/genetics/metabolism
Hemolysin Proteins/genetics/metabolism
Insecticide Resistance/genetics

@article {pmid38490212,
year = {2024},
author = {Lin, CP and Levy, PL and Alflen, A and Apriamashvili, G and Ligtenberg, MA and Vredevoogd, DW and Bleijerveld, OB and Alkan, F and Malka, Y and Hoekman, L and Markovits, E and George, A and Traets, JJH and Krijgsman, O and van Vliet, A and Poźniak, J and Pulido-Vicuña, CA and de Bruijn, B and van Hal-van Veen, SE and Boshuizen, J and van der Helm, PW and Díaz-Gómez, J and Warda, H and Behrens, LM and Mardesic, P and Dehni, B and Visser, NL and Marine, JC and Markel, G and Faller, WJ and Altelaar, M and Agami, R and Besser, MJ and Peeper, DS},
title = {Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness.},
journal = {Cancer cell},
volume = {42},
number = {4},
pages = {623-645.e10},
pmid = {38490212},
issn = {1878-3686},
mesh = {Humans ; *CRISPR-Cas Systems ; CD8-Positive T-Lymphocytes ; *Neoplasms/genetics ; },
abstract = {Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.},
}

Humans
*CRISPR-Cas Systems
CD8-Positive T-Lymphocytes
*Neoplasms/genetics

@article {pmid38462771,
year = {2024},
author = {Li, M and Zhang, CL and Zhou, DS and Chan, SH and Liu, XQ and Chen, SN and Yang, ZY and Ju, FE and Sang, XY and Liu, ZX and Zhang, QX and Pan, YM and Deng, SS and Wang, XM and Zhong, L and Zhang, XD and Du, X},
title = {Identification of COQ2 as a regulator of proliferation and lipid peroxidation through genome-scale CRISPR-Cas9 screening in myeloma cells.},
journal = {British journal of haematology},
volume = {204},
number = {4},
pages = {1307-1324},
doi = {10.1111/bjh.19375},
pmid = {38462771},
issn = {1365-2141},
support = {32100564//National Nature Science Foundation of China/ ; 81970191//National Nature Science Foundation of China/ ; 82002899//National Nature Science Foundation of China/ ; 82373026//National Nature Science Foundation of China/ ; JCYJ20190807160209294//Shenzhen Science and Technology Program/ ; JCYJ20220530144607017//Shenzhen Science and Technology Program/ ; 2022A1515220110//Guangdong Basic and Applied Basic Research Foundation/ ; 2023A1515012662//Guangdong Basic and Applied Basic Research Foundation/ ; A2303011//Shenzhen Medical Research Funds (SMART)/ ; 2021B1212040006//Science and Technology Planning Project of Guangdong Province/ ; SZXK008//Shenzhen Key Medical Discipline Construction Fund/ ; },
mesh = {Humans ; Animals ; Mice ; *Multiple Myeloma/drug therapy ; CRISPR-Cas Systems ; Lipid Peroxidation ; Mice, Inbred CBA ; Mice, Knockout ; Neoplasm Recurrence, Local ; Cell Proliferation ; Disease Models, Animal ; Mitogen-Activated Protein Kinase Kinases/therapeutic use ; Cell Line, Tumor ; },
abstract = {Multiple myeloma (MM) is the second most common malignant haematological disease with a poor prognosis. The limit therapeutic progress has been made in MM patients with cancer relapse, necessitating deeper research into the molecular mechanisms underlying its occurrence and development. A genome-wide CRISPR-Cas9 loss-of-function screening was utilized to identify potential therapeutic targets in our research. We revealed that COQ2 plays a crucial role in regulating MM cell proliferation and lipid peroxidation (LPO). Knockout of COQ2 inhibited cell proliferation, induced cell cycle arrest and reduced tumour growth in vivo. Mechanistically, COQ2 promoted the activation of the MEK/ERK cascade, which in turn stabilized and activated MYC protein. Moreover, we found that COQ2-deficient MM cells increased sensitivity to the LPO activator, RSL3. Using an inhibitor targeting COQ2 by 4-CBA enhanced the sensitivity to RSL3 in primary CD138[+] myeloma cells and in a xenograft mouse model. Nevertheless, co-treatment of 4-CBA and RSL3 induced cell death in bortezomib-resistant MM cells. Together, our findings suggest that COQ2 promotes cell proliferation and tumour growth through the activation of the MEK/ERK/MYC axis and targeting COQ2 could enhance the sensitivity to ferroptosis in MM cells, which may be a promising therapeutic strategy for the treatment of MM patients.},
}

Humans
Animals
Mice
*Multiple Myeloma/drug therapy
CRISPR-Cas Systems
Lipid Peroxidation
Mice, Inbred CBA
Mice, Knockout
Neoplasm Recurrence, Local
Cell Proliferation
Disease Models, Animal
Mitogen-Activated Protein Kinase Kinases/therapeutic use
Cell Line, Tumor

@article {pmid38418802,
year = {2024},
author = {Stojak, J and Rocha, D and Mörke, C and Kühn, C and Blanquet, V and Taniguchi, H},
title = {Establishment of a cloning-free CRISPR/Cas9 protocol to generate large deletions in the bovine MDBK cell line.},
journal = {Journal of applied genetics},
volume = {65},
number = {2},
pages = {399-402},
pmid = {38418802},
issn = {2190-3883},
support = {815668//H2020 European Research Council/ ; },
mesh = {Cattle ; Animals ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Base Sequence ; Cell Line ; },
abstract = {The CRISPR/Cas9 technique applied to modify the cattle genome has value in increasing animal health and welfare. Here, we established a simple, fast, and efficient cloning-free CRISPR/Cas9 protocol for large deletions of genomic loci in the frequently used model bovine MDBK cell line. The main advantages of our protocol are as follows: (i) pre-screening of the sgRNA efficiency with a fast and simple cleavage assay, (ii) reliable detection of genomic edits primarily by PCR and confirmed by DNA sequencing, and (iii) single cell sorting with FACS providing specific genetic information from modified cells of interest. Therefore, our method could be successfully applied in different studies, including functional validation of any genetic or regulatory elements.},
}

Cattle
Animals
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems
Base Sequence
Cell Line

@article {pmid38411080,
year = {2024},
author = {Rathore, U and Haas, P and Easwar Kumar, V and Hiatt, J and Haas, KM and Bouhaddou, M and Swaney, DL and Stevenson, E and Zuliani-Alvarez, L and McGregor, MJ and Turner-Groth, A and Ochieng' Olwal, C and Bediako, Y and Braberg, H and Soucheray, M and Ott, M and Eckhardt, M and Hultquist, JF and Marson, A and Kaake, RM and Krogan, NJ},
title = {CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.},
journal = {mBio},
volume = {15},
number = {4},
pages = {e0222223},
pmid = {38411080},
issn = {2150-7511},
support = {P50 AI150476, R01 AI120694, P01 AI063302//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 1110189-69-RKRL//amfAR, The Foundation for AIDS Research (amfAR)/ ; DP2 DA042423-01//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; H22BD4486//California HIV/AIDS Research Program (CHRP)/ ; },
mesh = {Humans ; *HIV Infections ; TNF Receptor-Associated Factor 2/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; Virus Latency ; CRISPR-Cas Systems ; Virus Replication ; CD4-Positive T-Lymphocytes ; Ubiquitins/metabolism ; CCAAT-Enhancer-Binding Proteins/genetics/metabolism ; },
abstract = {UNLABELLED: During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies.

IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.},
}

Humans
*HIV Infections
TNF Receptor-Associated Factor 2/metabolism
Ubiquitin-Protein Ligases/genetics/metabolism
Virus Latency
CRISPR-Cas Systems
Virus Replication
CD4-Positive T-Lymphocytes
Ubiquitins/metabolism
CCAAT-Enhancer-Binding Proteins/genetics/metabolism

@article {pmid38351739,
year = {2024},
author = {Zhao, S and Han, X and Zhu, Y and Han, Y and Liu, H and Chen, Z and Li, H and Wang, D and Tian, C and Yuan, Y and Guo, Y and Si, X and Wang, D and Ji, X},
title = {CRISPR/CasΦ2-mediated gene editing in wheat and rye.},
journal = {Journal of integrative plant biology},
volume = {66},
number = {4},
pages = {638-641},
doi = {10.1111/jipb.13624},
pmid = {38351739},
issn = {1744-7909},
support = {32000286//National Natural Science Foundation of China/ ; 32370432//National Natural Science Foundation of China/ ; 2021YFF1000203//National Key Research and Development Program of China/ ; },
mesh = {*Gene Editing ; *Triticum/genetics ; CRISPR-Cas Systems/genetics ; Secale/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The compact CRISPR/CasΦ2 system provides a complementary genome engineering tool for efficient gene editing including cytosine and adenosine base editing in wheat and rye with high specificity, efficient use of the protospacer-adjacent motif TTN, and an alternative base-editing window.},
}

*Gene Editing
*Triticum/genetics
CRISPR-Cas Systems/genetics
Secale/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid38320883,
year = {2024},
author = {Yong, J and Wu, M and Carroll, BJ and Xu, ZP and Zhang, R},
title = {Enhancing plant biotechnology by nanoparticle delivery of nucleic acids.},
journal = {Trends in genetics : TIG},
volume = {40},
number = {4},
pages = {352-363},
doi = {10.1016/j.tig.2024.01.005},
pmid = {38320883},
issn = {0168-9525},
mesh = {*Nucleic Acids ; CRISPR-Cas Systems ; Genome, Plant ; Plants/genetics ; Biotechnology ; Gene Editing ; *Nanoparticles ; Plants, Genetically Modified/genetics ; },
abstract = {Plant biotechnology plays a crucial role in developing modern agriculture and plant science research. However, the delivery of exogenous genetic material into plants has been a long-standing obstacle. Nanoparticle-based delivery systems are being established to address this limitation and are proving to be a feasible, versatile, and efficient approach to facilitate the internalization of functional RNA and DNA by plants. The nanoparticle-based delivery systems can also be designed for subcellular delivery and controlled release of the biomolecular cargo. In this review, we provide a concise overview of the recent advances in nanocarriers for the delivery of biomolecules into plants, with a specific focus on applications to enhance RNA interference, foreign gene transfer, and genome editing in plants.},
}

*Nucleic Acids
CRISPR-Cas Systems
Genome, Plant
Plants/genetics
Biotechnology
Gene Editing
*Nanoparticles
Plants, Genetically Modified/genetics

@article {pmid38217286,
year = {2024},
author = {Ma, G and Yan, F and Ren, B and Lu, Z and Xu, H and Wu, F and Li, S and Wang, D and Zhou, X and Zhou, H},
title = {LbCas12a-nuclease-mediated tiling deletion for large-scale targeted editing of non-coding regions in rice.},
journal = {Plant communications},
volume = {5},
number = {4},
pages = {100815},
doi = {10.1016/j.xplc.2024.100815},
pmid = {38217286},
issn = {2590-3462},
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
}

*Oryza/genetics
CRISPR-Cas Systems/genetics
Gene Editing

@article {pmid38571947,
year = {2024},
author = {Yang, Y and Xu, Z and Tao, Q and Xu, L and Gu, S and Huang, Y and Liu, Z and Zhang, Y and Wen, J and Lai, S and Zhu, L},
title = {Construction of recombinant pseudorabies virus expressing PCV2 Cap, PCV3 Cap, and IL-4: investigation of their biological characteristics and immunogenicity.},
journal = {Frontiers in immunology},
volume = {15},
number = {},
pages = {1339387},
pmid = {38571947},
issn = {1664-3224},
mesh = {Swine ; Animals ; Mice ; *Herpesvirus 1, Suid/genetics ; *Pseudorabies/prevention & control ; Interleukin-4/genetics ; *Circovirus/genetics ; Vaccines, Synthetic ; },
abstract = {BACKGROUND: Porcine circovirus type 2 (PCV2) is a globally prevalent and recurrent pathogen that primarily causes slow growth and immunosuppression in pigs. Porcine circovirus type 3 (PCV3), a recently discovered virus, commonly leads to reproductive disorders in pigs and has been extensively disseminated worldwide. Infection with a single PCV subtype alone does not induce severe porcine circovirus-associated diseases (PCVD), whereas concurrent co-infection with PCV2 and PCV3 exacerbates the clinical manifestations. Pseudorabies (PR), a highly contagious disease in pigs, pose a significant threat to the swine industry in China.

METHODS: In this study, recombinant strains named rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 was constructed by using a variant strain XJ of pseudorabies virus (PRV) as the parental strain, with the TK/gE/gI genes deleted and simultaneous expression of PCV2 Cap, PCV3 Cap, and IL-4. The two recombinant strains obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster Syrian kidney-21 (BHK-21) cells and is safe to mice.

RESULTS: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 exhibited good safety and immunogenicity in mice, inducing high levels of antibodies, demonstrated 100% protection against the PRV challenge in mice, reduced viral loads and mitigated pathological changes in the heart, lungs, spleen, and lymph nodes during PCV2 challenge. Moreover, the recombinant viruses with the addition of IL-4 as a molecular adjuvant outperformed the non-addition group in most indicators.

CONCLUSION: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 hold promise as recombinant vaccines for the simultaneous prevention of PCV2, PCV3, and PRV, while IL-4, as a vaccine molecular adjuvant, effectively enhances the immune response of the vaccine.},
}

Swine
Animals
Mice
*Herpesvirus 1, Suid/genetics
*Pseudorabies/prevention & control
Interleukin-4/genetics
*Circovirus/genetics
Vaccines, Synthetic

@article {pmid38570691,
year = {2024},
author = {Yan, J and Oyler-Castrillo, P and Ravisankar, P and Ward, CC and Levesque, S and Jing, Y and Simpson, D and Zhao, A and Li, H and Yan, W and Goudy, L and Schmidt, R and Solley, SC and Gilbert, LA and Chan, MM and Bauer, DE and Marson, A and Parsons, LR and Adamson, B},
title = {Improving prime editing with an endogenous small RNA-binding protein.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {38570691},
issn = {1476-4687},
abstract = {Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs[1]. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts[2]. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.},
}

@article {pmid38570576,
year = {2024},
author = {Sutipatanasomboon, A and Wongsantichon, J and Sakdee, S and Naksith, P and Watthanadirek, A and Anuracpreeda, P and Blacksell, SD and Saisawang, C},
title = {RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {7820},
pmid = {38570576},
issn = {2045-2322},
support = {/WT_/Wellcome Trust/United Kingdom ; 220211/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Cattle ; Animals ; *Anaplasma marginale/genetics ; *Anaplasmosis/diagnosis/genetics ; CRISPR-Cas Systems ; *Cattle Diseases/genetics ; *Tick-Borne Diseases/genetics ; },
abstract = {Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/μl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines.},
}

Cattle
Animals
*Anaplasma marginale/genetics
*Anaplasmosis/diagnosis/genetics
CRISPR-Cas Systems
*Cattle Diseases/genetics
*Tick-Borne Diseases/genetics

@article {pmid38567981,
year = {2024},
author = {Safarkhani, M and Farasati Far, B and Kim, SH and Makvandi, P and Park, MK and Huh, Y and Rabiee, N},
title = {Advances and Challenges of Sensing in Water Using CRISPR-Cas Technology.},
journal = {ACS biomaterials science & engineering},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsbiomaterials.3c01689},
pmid = {38567981},
issn = {2373-9878},
abstract = {The groundbreaking gene-editing mechanism, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), paired with the protein Cas9, has significantly advanced the realms of biology, medicine, and agriculture. Through its precision in modifying genetic sequences, CRISPR holds the potential to alter the trajectory of genetic disorders and accelerate advancements in agriculture. While its therapeutic potential is profound, the technology also invites ethical debates centered on responsible use and equity in access. Parallelly, in the environmental monitoring sphere and sensing in water, especially biosensors have been instrumental in evaluating natural water sources' quality. These biosensors, integrating biological components with detection techniques, have the potential to revolutionize healthcare by providing rapid and minimally invasive diagnostic methods. However, the design and application of these sensors bring forth challenges, especially in ensuring sensitivity, selectivity, and ethical data handling. This article delves into the prospective use of CRISPR-Cas technology for sensing in water, exploring its capabilities in detecting diverse biomarkers, hazardous substances, and varied reactions in water and wastewater systems.},
}

@article {pmid38567969,
year = {2024},
author = {Maes, A and Botzki, A and Mathys, J and Impens, F and Saelens, X},
title = {Systematic review and meta-analysis of genome-wide pooled CRISPR screens to identify host factors involved in influenza A virus infection.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0185723},
doi = {10.1128/jvi.01857-23},
pmid = {38567969},
issn = {1098-5514},
abstract = {UNLABELLED: The host-virus interactome is increasingly recognized as an important research field to discover new therapeutic targets to treat influenza. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify new pro- and antiviral host factors of the influenza A virus. However, at present, a comprehensive summary of the results is lacking. We performed a systematic review of all reported CRISPR studies in this field in combination with a meta-analysis using the algorithm of meta-analysis by information content (MAIC). Two ranked gene lists were generated based on evidence in 15 proviral and 4 antiviral screens. Enriched pathways in the proviral MAIC results were compared to those of a prior array-based RNA interference (RNAi) meta-analysis. The top 50 proviral MAIC list contained genes whose role requires further elucidation, such as the endosomal ion channel TPCN1 and the kinase WEE1. Moreover, MAIC indicated that ALYREF, a component of the transcription export complex, has antiviral properties, whereas former knockdown experiments attributed a proviral role to this host factor. CRISPR-Cas-pooled screens displayed a bias toward early-replication events, whereas the prior RNAi meta-analysis covered early and late-stage events. RNAi screens led to the identification of a larger fraction of essential genes than CRISPR screens. In summary, the MAIC algorithm points toward the importance of several less well-known pathways in host-influenza virus interactions that merit further investigation. The results from this meta-analysis of CRISPR screens in influenza A virus infection may help guide future research efforts to develop host-directed anti-influenza drugs.

IMPORTANCE: Viruses rely on host factors for their replication, whereas the host cell has evolved virus restriction factors. These factors represent potential targets for host-oriented antiviral therapies. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify pro- and antiviral host factors in the context of influenza virus infection. We performed a comprehensive analysis of the outcome of these screens based on the publicly available gene lists, using the recently developed algorithm meta-analysis by information content (MAIC). MAIC allows the systematic integration of ranked and unranked gene lists into a final ranked gene list. This approach highlighted poorly characterized host factors and pathways with evidence from multiple screens, such as the vesicle docking and lipid metabolism pathways, which merit further exploration.},
}

@article {pmid38567606,
year = {2024},
author = {Saifullah, M and Laghzaoui, O and Ozyahyalar, H and Irfan, A},
title = {The CRISPR-Cas9 induced CCR5 Δ32 mutation as a potent gene therapy methodology for resistance to HIV-1 variant: a review.},
journal = {European review for medical and pharmacological sciences},
volume = {28},
number = {6},
pages = {2430-2463},
doi = {10.26355/eurrev_202403_35751},
pmid = {38567606},
issn = {2284-0729},
mesh = {Humans ; *HIV-1/genetics ; *Acquired Immunodeficiency Syndrome/genetics/therapy ; *HIV Infections/genetics/therapy ; CRISPR-Cas Systems/genetics ; Receptors, CCR5/genetics/metabolism ; Mutation ; Genetic Therapy ; Polymorphism, Single Nucleotide ; Gene Frequency ; },
abstract = {Human Immunodeficiency Virus (HIV) has continuously been the greatest epidemic for humanity over a period spanning almost five decades. With no specific cure or treatment available to date despite extensive research, the C-C Chemokine Receptor 5, Delta 32 (CCR5 Δ32) allele genetic point mutation plays an imperative role in the prevention of acquired immunodeficiency syndrome (AIDS). This comprehensive study aims to review the induction of the homozygous recessive deletion genotype using the Clustered Regularly Interspaced Short Palindromic Repeats, Cas 9 Enzyme (CRISPR-Cas9), and hematopoietic stem cell transplantation under positive selection pressure for active immunity in seropositive patients' populations as the phenotype. A methodology is proposed to trigger a significant increase in the expression of Delta 32 beneficial mutant alleles within controlled modern healthcare facilities utilizing totipotent stem cells through somatic gene therapy. It acts upon two dysfunctional CCR5 genes, translating mutant G protein-coupled co-receptors, whose primary function is similar to that of C-X-C Motif Chemokine receptor 4 (CXCR4), by blocking the entry of viral RNA into the CD4+ T helper lymphocytes, halting infection and seizing viral life cycle. This modification is endemic in Northern Europe, where it naturally pertains to the Caucasian descent population samples in the form of polymorphism, p (X=0.01), where X is the probability of frequency of complete immunity against HIV-1 in population samples. The epigenetics of the single nucleotide polymorphism (SNP) are analyzed as they play a significant role in immunity distribution. Furthermore, a comparative analysis within the ethical boundaries of CRISPR-Cas9 is conducted to discuss the practical aspects and challenges of the presented methodologies and treatment alternatives. Additionally, the study assembles all available data and summarizes preexisting research while providing a promising solution to this ethical dilemma. Finally, a methodology is devised to answer the question of whether the variant-specific epidemic of AIDS caused by HIV-1 can be cured via artificially inducing immunity by CRISPR-Cas9.},
}

Humans
*HIV-1/genetics
*Acquired Immunodeficiency Syndrome/genetics/therapy
*HIV Infections/genetics/therapy
CRISPR-Cas Systems/genetics
Receptors, CCR5/genetics/metabolism
Mutation
Genetic Therapy
Polymorphism, Single Nucleotide
Gene Frequency

@article {pmid38509377,
year = {2024},
author = {Lee, HG and Rone, JM and Li, Z and Akl, CF and Shin, SW and Lee, JH and Flausino, LE and Pernin, F and Chao, CC and Kleemann, KL and Srun, L and Illouz, T and Giovannoni, F and Charabati, M and Sanmarco, LM and Kenison, JE and Piester, G and Zandee, SEJ and Antel, JP and Rothhammer, V and Wheeler, MA and Prat, A and Clark, IC and Quintana, FJ},
title = {Disease-associated astrocyte epigenetic memory promotes CNS pathology.},
journal = {Nature},
volume = {627},
number = {8005},
pages = {865-872},
pmid = {38509377},
issn = {1476-4687},
support = {R00 NS114111/NS/NINDS NIH HHS/United States ; R01 MH130458/MH/NIMH NIH HHS/United States ; R01 MH132632/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Female ; Humans ; Male ; Mice ; Acetyl Coenzyme A/metabolism ; *Astrocytes/enzymology/metabolism/pathology ; ATP Citrate (pro-S)-Lyase/metabolism ; Chromatin/genetics/metabolism ; Chromatin Assembly and Disassembly ; Chromatin Immunoprecipitation Sequencing ; CRISPR-Cas Systems ; *Encephalomyelitis, Autoimmune, Experimental/enzymology/genetics/metabolism/pathology ; *Epigenetic Memory ; Inflammation/enzymology/genetics/metabolism/pathology ; *Multiple Sclerosis/enzymology/genetics/metabolism/pathology ; Single-Cell Gene Expression Analysis ; Transposases/metabolism ; },
abstract = {Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis[1-8] (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY[+]p300[+] memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY[+]p300[+] astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.},
}

Animals
Female
Humans
Male
Mice
Acetyl Coenzyme A/metabolism
*Astrocytes/enzymology/metabolism/pathology
ATP Citrate (pro-S)-Lyase/metabolism
Chromatin/genetics/metabolism
Chromatin Assembly and Disassembly
Chromatin Immunoprecipitation Sequencing
CRISPR-Cas Systems
*Encephalomyelitis, Autoimmune, Experimental/enzymology/genetics/metabolism/pathology
*Epigenetic Memory
Inflammation/enzymology/genetics/metabolism/pathology
*Multiple Sclerosis/enzymology/genetics/metabolism/pathology
Single-Cell Gene Expression Analysis
Transposases/metabolism

@article {pmid38401849,
year = {2024},
author = {Wang, X and Li, Y and A, S and Lyu, J and Wang, X and He, Z and Lara-Sáez, I and Li, M and Wang, W},
title = {Cyclization-enhanced poly(β-amino ester)s vectors for efficient CRISPR gene editing therapy.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {368},
number = {},
pages = {444-452},
doi = {10.1016/j.jconrel.2024.02.032},
pmid = {38401849},
issn = {1873-4995},
mesh = {Humans ; *Gene Editing/methods ; Cyclization ; *Esters ; RNA, Guide, CRISPR-Cas Systems ; DNA/metabolism ; CRISPR-Cas Systems/genetics ; Collagen Type VII/genetics ; *Polymers ; },
abstract = {Among non-viral gene delivery vectors, poly(β-amino ester)s (PAEs) are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. Over two decades, PAEs have evolved from linear to highly branched structures, significantly enhancing gene delivery efficacy. Building on the proven efficient sets of monomers in highly branched PAEs (HPAEs), this work introduced a new class of cyclic PAEs (CPAEs) constructed via an A2 + B4 + C2 cyclization synthesis strategy and identified their markedly improved gene transfection capabilities in gene delivery applications. Two sets of cyclic PAEs (CPAEs) with rings of different sizes and topologies were obtained. Their chemical structures were confirmed via two-dimensional nuclear magnetic resonance and the photoluminescence phenomena, and their DNA delivery behaviours were investigated and compared with the HPAE counterparts. In vitro assessments demonstrated that the CPAEs with a macrocyclic architecture (MCPAEs), significantly enhanced DNA intracellular uptake and facilitated efficient gene expression while maintaining perfect biocompatibility. The top-performance MCPAEs have been further employed to deliver a plasmid coding dual single guide RNA-guided CRISPR-Cas9 machinery to delete COL7A1 exon 80 containing the c.6527dupC mutation. In recessive dystrophic epidermolysis bullosa (RDEB) patient-derived epidermal keratinocytes, MCPAEs facilitated the CRISPR plasmid delivery and achieved efficient targeted gene editing in multiple colonies.},
}

Humans
*Gene Editing/methods
Cyclization
*Esters
RNA, Guide, CRISPR-Cas Systems
DNA/metabolism
CRISPR-Cas Systems/genetics
Collagen Type VII/genetics
*Polymers

@article {pmid38199433,
year = {2024},
author = {Chen, J and Qin, H and Hao, J and Wang, Q and Chen, S and Yang, G and Li, M and Zhu, X and Wang, D and Chen, H and Cui, C and Chen, M},
title = {Cardiac-specific overexpression of CREM-IbΔC-X via CRISPR/Cas9 in mice presents a new model of atrial cardiomyopathy with spontaneous atrial fibrillation.},
journal = {Translational research : the journal of laboratory and clinical medicine},
volume = {267},
number = {},
pages = {54-66},
doi = {10.1016/j.trsl.2024.01.001},
pmid = {38199433},
issn = {1878-1810},
mesh = {Mice ; Humans ; Animals ; *Atrial Fibrillation ; CRISPR-Cas Systems/genetics ; Mice, Transgenic ; Heart Atria/pathology ; *Cardiomyopathies/genetics ; Cyclic AMP Response Element Modulator/genetics/metabolism ; },
abstract = {Atrial cardiomyopathy (ACM) forms the substrate for atrial fibrillation (AF) and underlies the potential for atrial thrombus formation and subsequent stroke. However, generating stable animal models that accurately replicate the entire progression of atrial lesions, particularly the onset of AF, presents significant challenges. In the present study, we found that the isoform of CRE-binding protein modulator (CREM-IbΔC-X), which is involved in the regulation of cardiac development and atrial rhythm, was highly expressed in atrial biopsies from patients with AF. Building upon this finding, we employed CRISPR/Cas9 technology to create a mouse model with cardiac-specific overexpression of CREM-IbΔC-X (referred to as CS-CREM mice). This animal model effectively illustrated the development of ACM through electrophysiological and structural remodelings over time. Proteomics and Chip-qPCR analysis of atrial samples revealed significant upregulation of cell-matrix adhesion and extracellular matrix structural components, alongside significant downregulation of genes related to atrial functions in the CS-CREM mice. Furthermore, the corresponding responses to anti-arrhythmia drugs, i.e., amiodarone and propafenone, suggested that CS-CREM mice could serve as an ideal in vivo model for drug testing. Our study introduced a novel ACM model with spontaneous AF by cardiac-specifically overexpressing CREM-IbΔC-X in mice, providing valuable insights into the mechanisms and therapeutic targets of ACM.},
}

Mice
Humans
Animals
*Atrial Fibrillation
CRISPR-Cas Systems/genetics
Mice, Transgenic
Heart Atria/pathology
*Cardiomyopathies/genetics
Cyclic AMP Response Element Modulator/genetics/metabolism

@article {pmid38521024,
year = {2024},
author = {Zhu, L and Zhang, X and Yang, L and Qiu, S and Liu, G and Xiong, X and Xiao, T and Huang, K and Zhu, L},
title = {Label-free electrochemical sensing platform for sensitive detection of ampicillin by combining nucleic acid isothermal enzyme-free amplification circuits with CRISPR/Cas12a.},
journal = {Talanta},
volume = {273},
number = {},
pages = {125950},
doi = {10.1016/j.talanta.2024.125950},
pmid = {38521024},
issn = {1873-3573},
mesh = {Humans ; *Nucleic Acids ; CRISPR-Cas Systems ; Nucleic Acid Hybridization ; Ampicillin ; *Biosensing Techniques/methods ; },
abstract = {The residue of ampicillin (AMP) in food and ecological environment poses a potential harm to human health. Therefore, a reliable system for detecting AMP is in great demand. Herein, a label-free and sensitive electrochemical sensor utilizing NH2-Co-MOF as an electrocatalytic active material for methylene blue (MB) was developed for rapid and facile AMP detection by combining hybridization chain reaction (HCR), catalytic hairpin assembly (CHA) with CRISPR/Cas12a. The surface of glassy carbon electrode modified with NH2-Co-MOF was able to undergo HCR independent of the AMP, forming long dsDNA complexes to load MB, resulting in strong original electrochemical signal. The presence of AMP could trigger upstream CHA circuit to activate the CRISPR/Cas12a system, thereby achieving rapid non-specific cleavage of the trigger ssDNA of HCR on the electrode surface, hindering the occurrence of HCR and reducing the load of MB. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the AMP with a detection limit as low as 1.60 pM (S/N = 3). The proposed sensor exhibited good stability, selectivity, and stability, and achieved reliable detection of AMP in milk and livestock wastewater samples, demonstrating its promising application prospects in food safety and environmental monitoring.},
}

Humans
*Nucleic Acids
CRISPR-Cas Systems
Nucleic Acid Hybridization
Ampicillin
*Biosensing Techniques/methods

@article {pmid38503125,
year = {2024},
author = {Peng, J and Liu, T and Guan, L and Xu, Z and Xiong, T and Zhang, Y and Song, J and Liu, X and Yang, Y and Hao, X},
title = {A highly sensitive Lock-Cas12a biosensor for detection and imaging of miRNA-21 in breast cancer cells.},
journal = {Talanta},
volume = {273},
number = {},
pages = {125938},
doi = {10.1016/j.talanta.2024.125938},
pmid = {38503125},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics ; CRISPR-Cas Systems ; Diagnostic Imaging ; Limit of Detection ; *Nucleic Acids ; *Biosensing Techniques ; *Neoplasms ; },
abstract = {The expression levels of microRNA (miRNA) vary significantly in correlation with the occurrence and progression of cancer, making them valuable biomarkers for cancer diagnosis. However, their quantitative detection faces challenges due to the high sequence homology, low abundance and small size. In this work, we established a strand displacement amplification (SDA) approach based on miRNA-triggered structural "Lock" nucleic acid ("Lock" DNA), coupled with the CRISPR/Cas12a system, for detecting miRNA-21 in breast cancer cells. The "Lock" DNA freed the CRISPR-derived RNA (crRNA) from the dependence on the target sequence and greatly facilitated the extended detection of different miRNAs. Moreover, the CRISPR/Cas12a system provided excellent amplification ability and specificity. The designed biosensor achieved high sensitivity detection of miRNA-21 with a limit of detection (LOD) of 28.8 aM. In particular, the biosensor could distinguish breast cancer cells from other cancer cells through intracellular imaging. With its straightforward sequence design and ease of use, the Lock-Cas12a biosensor offers significant advantages for cell imaging and early clinical diagnosis.},
}

*MicroRNAs/genetics
CRISPR-Cas Systems
Diagnostic Imaging
Limit of Detection
*Nucleic Acids
*Biosensing Techniques
*Neoplasms

@article {pmid38503121,
year = {2024},
author = {Li, T and Wang, J and Fang, J and Chen, F and Wu, X and Wang, L and Gao, M and Zhang, L and Li, S},
title = {A universal nucleic acid detection platform combing CRISPR/Cas12a and strand displacement amplification with multiple signal readout.},
journal = {Talanta},
volume = {273},
number = {},
pages = {125922},
doi = {10.1016/j.talanta.2024.125922},
pmid = {38503121},
issn = {1873-3573},
mesh = {Male ; Humans ; CRISPR-Cas Systems ; Semen ; Biological Assay ; *Nucleic Acids ; DNA ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques ; },
abstract = {Rapid and sensitive detection of nucleic acids has become crucial in various fields. However, most current nucleic acid detection methods can only be used in specific scenarios, such as RT-qPCR, which relies on fluorometer for signal readout, limiting its application at home or in the field due to its high price. In this paper, a universal nucleic acid detection platform combing CRISPR/Cas12a and strand displacement amplification (CRISPR-SDA) with multiple signal readout was established to adapt to different application scenarios. Nucleocapsid protein gene of SARS-CoV-2 (N gene) and hepatitis B virus (HBV) DNA were selected as model targets. The proposed strategy achieved the sensitivity of 53.1 fM, 0.15 pM, and 1 pM for N gene in fluorescence mode, personal glucose meter (PGM) mode and lateral flow assay (LFA) mode, respectively. It possessed the ability to differentiate single-base mismatch and the presence of salmon sperm DNA with a mass up to 10[5]-fold of the targets did not significantly interfere with the assay signal. The general and modular design idea made CRISPR-SDA as simple as building blocks to construct nucleic acid sensing methods to meet different requirements by simply changing the SDA template and selecting suitable signal report probes, which was expected to find a breadth of applications in nucleic acids detection.},
}

Male
Humans
CRISPR-Cas Systems
Semen
Biological Assay
*Nucleic Acids
DNA
Nucleic Acid Amplification Techniques
*Biosensing Techniques

@article {pmid38432581,
year = {2024},
author = {Rodrigo-Faus, M and Vincelle-Nieto, A and Vidal, N and Puente, J and Saiz-Pardo, M and Lopez-Garcia, A and Mendiburu-Eliçabe, M and Palao, N and Baquero, C and Linzoain-Agos, P and Cuesta, AM and Qu, HQ and Hakonarson, H and Musteanu, M and Reyes-Palomares, A and Porras, A and Bragado, P and Gutierrez-Uzquiza, A},
title = {CRISPR/Cas9 screenings unearth protein arginine methyltransferase 7 as a novel essential gene in prostate cancer metastasis.},
journal = {Cancer letters},
volume = {588},
number = {},
pages = {216776},
doi = {10.1016/j.canlet.2024.216776},
pmid = {38432581},
issn = {1872-7980},
mesh = {Male ; Animals ; Mice ; Humans ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; *Prostatic Neoplasms, Castration-Resistant/pathology ; CRISPR-Cas Systems ; Genes, Essential ; Early Detection of Cancer ; },
abstract = {Due to the limited effectiveness of current treatments, the survival rate of patients with metastatic castration-resistant prostate cancer (mCRPC) is significantly reduced. Consequently, it is imperative to identify novel therapeutic targets for managing these patients. Since the invasive ability of cells is crucial for establishing and maintaining metastasis, the aim of this study was to identify the essential regulators of invasive abilities of mCRPC cells by conducting two independent high-throughput CRISPR/Cas9 screenings. Furthermore, some of the top hits were validated using siRNA technology, with protein arginine methyltransferase 7 (PRMT7) emerging as the most promising candidate. We demonstrated that its inhibition or depletion via genetic or pharmacological approaches significantly reduces invasive, migratory and proliferative abilities of mCRPC cells in vitro. Moreover, we confirmed that PRMT7 ablation reduces cell dissemination in chicken chorioallantoic membrane and mouse xenograft assays. Molecularly, PRMT7 reprograms the expression of several adhesion molecules by methylating various transcription factors, such as FoxK1, resulting in the loss of adhesion from the primary tumor and increased motility of mCRPC cells. Furthermore, PRMT7 higher expression correlates with tumor aggressivity and poor overall survival in prostate cancer patients. Thus, this study demonstrates that PRMT7 is a potential therapeutic target and potential biomarker for mPCa.},
}

Male
Animals
Mice
Humans
*Protein-Arginine N-Methyltransferases/genetics/metabolism
*Prostatic Neoplasms, Castration-Resistant/pathology
CRISPR-Cas Systems
Genes, Essential
Early Detection of Cancer

@article {pmid38331220,
year = {2024},
author = {Rong, F and Wang, H and Tang, X and Xing, J and Sheng, X and Chi, H and Zhan, W},
title = {The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus (IHNV).},
journal = {Journal of virological methods},
volume = {326},
number = {},
pages = {114892},
doi = {10.1016/j.jviromet.2024.114892},
pmid = {38331220},
issn = {1879-0984},
mesh = {Animals ; *Infectious hematopoietic necrosis virus/genetics ; CRISPR-Cas Systems ; Reverse Transcription ; Recombinases/genetics ; },
abstract = {Infectious hematopoietic necrosis virus (IHNV) is an economically important virus causing significant mortalities among wild and cultured salmonid fish worldwide. Rapid and sensitive diagnostic methods of IHNV are crucial for timely controlling infections. For better detection of IHNV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of IHNV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 41 °C and 35 min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15 min. The whole detection procedure including can be accomplished within one hour, with a detection sensitivity of about 9.5 copies/µL. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses HIRRV and VHSV, allowing naked-eye interpretation of the results through lateral flow or fluorescence under ultraviolet light. Overall, our results demonstrated that the developed RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for routine and on-site detection of IHNV, which shows a great application promise for the prevention of IHNV infections.},
}

Animals
*Infectious hematopoietic necrosis virus/genetics
CRISPR-Cas Systems
Reverse Transcription
Recombinases/genetics

@article {pmid38565780,
year = {2024},
author = {Ly, LK and Ho, TM and Bui, TP and Nguyen, LT and Phan, Q and Le, NT and Khuat, LTM and Le, LH and Chu, HH and Pham, NB and Do, PT},
title = {CRISPR/Cas9 targeted mutations of OsDSG1 gene enhanced salt tolerance in rice.},
journal = {Functional & integrative genomics},
volume = {24},
number = {2},
pages = {70},
pmid = {38565780},
issn = {1438-7948},
mesh = {*Salt Tolerance/genetics ; CRISPR-Cas Systems ; *Oryza/metabolism ; Salt Stress ; Mutation ; },
abstract = {Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.},
}

*Salt Tolerance/genetics
CRISPR-Cas Systems
*Oryza/metabolism
Salt Stress
Mutation

@article {pmid38565160,
year = {2024},
author = {Morimoto, K and Suzuki, H and Kuno, A and Daitoku, Y and Tanimoto, Y and Kato, K and Murata, K and Sugiyama, F and Mizuno, S},
title = {Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions.},
journal = {Open biology},
volume = {14},
number = {4},
pages = {240007},
pmid = {38565160},
issn = {2046-2441},
mesh = {Animals ; Mice ; *Gene Editing ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems ; Mutagenesis ; *MicroRNAs/genetics ; },
abstract = {Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.},
}

Animals
Mice
*Gene Editing
RNA, Guide, CRISPR-Cas Systems
CRISPR-Cas Systems
Mutagenesis
*MicroRNAs/genetics

@article {pmid38564697,
year = {2024},
author = {Han, X and Lu, M and Zhang, Y and Liu, X and Zhang, Q and Bai, X and Man, S and Zhao, L and Ma, L},
title = {A Thermostable Cas12b-Powered Bioassay Coupled with Loop-Mediated Isothermal Amplification in a Customized "One-Pot" Vessel for Visual, Rapid, Sensitive, and On-Site Detection of Genetically Modified Crops.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.4c01028},
pmid = {38564697},
issn = {1520-5118},
abstract = {Genetically modified crops (GMCs) have been discussed due to unknown safety, and thus, it is imperative to develop an effective detection technology. CRISPR/Cas is deemed a burgeoning technology for nucleic acid detection. Herein, we developed a novel detection method for the first time, which combined thermostable Cas12b with loop-mediated isothermal amplification (LAMP), to detect genetically modified (GM) soybeans in a customized one-pot vessel. In our method, LAMP-specific primers were used to amplify the cauliflower mosaic virus 35S promoter (CaMV35S) of the GM soybean samples. The corresponding amplicons activated the trans-cleavage activity of Cas12b, which resulted in the change of fluorescence intensity. The proposed bioassay was capable of detecting synthetic plasmid DNA samples down to 10 copies/μL, and as few as 0.05% transgenic contents could be detected in less than 40 min. This work presented an original detection method for GMCs, which performed rapid, on-site, and deployable detection.},
}

@article {pmid38563791,
year = {2024},
author = {Zheng, Z and Xu, L and Dou, H and Zhou, Y and Feng, X and He, X and Tian, Z and Song, L and Gao, Y and Mo, G and Hu, J and Zhao, H and Wei, H and Church, GM and Yang, L},
title = {Testing multiplexed anti-ASFV CRISPR-Cas9 in reducing African swine fever virus.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0216423},
doi = {10.1128/spectrum.02164-23},
pmid = {38563791},
issn = {2165-0497},
abstract = {UNLABELLED: African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies.

IMPORTANCE: ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.},
}

@article {pmid38563119,
year = {2024},
author = {Jia, HY and Zhang, XY and Ye, BC and Yin, BC},
title = {An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.3c05975},
pmid = {38563119},
issn = {1520-6882},
abstract = {CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAM-presenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates. We demonstrated the feasibility and specificity of this system for specifically visualizing endogenous mRNA. By leveraging dCas12a-mediated precursor CRISPR RNA (pre-crRNA) processing and the orthogonality of dCas12a from different bacteria, we further demonstrated the proposed system as a simple and versatile molecular toolkit for multiplexed imaging of different types of RNA transcripts in live cells with high specificity. This programmable dCas12a system not only broadens the RNA imaging toolbox but also facilitates diverse applications for RNA manipulation.},
}

@article {pmid38561518,
year = {2024},
author = {Özcan, A and Yıbar, A and Kiraz, D and Ilıkkan, ÖK},
title = {Comprehensive analysis of the CRISPR-Cas systems in Streptococcus thermophilus strains isolated from traditional yogurts.},
journal = {Antonie van Leeuwenhoek},
volume = {117},
number = {1},
pages = {63},
pmid = {38561518},
issn = {1572-9699},
mesh = {*Streptococcus thermophilus/genetics ; CRISPR-Cas Systems ; Yogurt ; *Bacteriophages/genetics ; Plasmids/genetics ; },
abstract = {Phage resistance is crucial for lactic acid bacteria in the dairy industry. However, identifying all phages affecting these bacteria is challenging. CRISPR-Cas systems offer a resistance mechanism developed by bacteria and archaea against phages and plasmids. In this study, 11 S. thermophilus strains from traditional yogurts underwent analysis using next-generation sequencing (NGS) and bioinformatics tools. Initial characterization involved molecular ribotyping. Bioinformatics analysis of the NGS raw data revealed that all 11 strains possessed at least one CRISPR type. A total of 21 CRISPR loci were identified, belonging to CRISPR types II-A, II-C, and III-A, including 13 Type II-A, 1 Type III-C, and 7 Type III-A CRISPR types. By analyzing spacer sequences in S. thermophilus bacterial genomes and matching them with phage/plasmid genomes, notable strains emerged. SY9 showed prominence with 132 phage matches and 30 plasmid matches, followed by SY12 with 35 phage matches and 25 plasmid matches, and SY18 with 49 phage matches and 13 plasmid matches. These findings indicate the potential of S. thermophilus strains in phage/plasmid resistance for selecting starter cultures, ultimately improving the quality and quantity of dairy products. Nevertheless, further research is required to validate these results and explore the practical applications of this approach.},
}

*Streptococcus thermophilus/genetics
CRISPR-Cas Systems
Yogurt
*Bacteriophages/genetics
Plasmids/genetics