@article {pmid33472057,
year = {2021},
author = {Wang, B and Zhang, T and Yin, J and Yu, Y and Xu, W and Ding, J and Patel, DJ and Yang, H},
title = {Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2020.12.033},
pmid = {33472057},
issn = {1097-4164},
abstract = {Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.},
}
@article {pmid33471332,
year = {2021},
author = {Tang, X and Qi, Y and Zhang, Y},
title = {Single Transcript Unit CRISPR 2.0 Systems for Genome Editing in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {193-204},
pmid = {33471332},
issn = {1940-6029},
abstract = {CRISPR-Cas9 and Cas12a (formerly Cpf1), RNA-guided DNA endonucleases found from adaptive immune system in prokaryotes, have been engineered and widely adopted as two of the most powerful genome editing systems in plants. Recently, we developed a single transcript unit (STU) CRISPR 2.0 toolbox for applications in plants, which contains two STU-Cas9 systems and one STU-Cas12a system. Here, we describe a detailed protocol about using the STU CRISPR 2.0 systems to achieve single and multiplex genome editing in rice.},
}
@article {pmid33471330,
year = {2021},
author = {Liu, G and Qi, Y and Zhang, T},
title = {Analysis of Off-Target Mutations in CRISPR-Edited Rice Plants Using Whole-Genome Sequencing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {145-172},
pmid = {33471330},
issn = {1940-6029},
abstract = {The CRISPR/Cas systems have become the most widely used tool for genome editing in plants and beyond. However, CRISPR/Cas systems may cause unexpected off-target mutations due to sgRNA recognizing highly homologous DNA sequence elsewhere in the genome. Whole-genome sequencing (WGS) can be used to identify on- and off-target mutation. Here, we describe a pipeline of analyzing WGS data using a series of open source software for analysis of off-target mutations in CRISPR-edited rice plants. In this pipeline, the adapter is trimmed using SKEWER. Then, the cleaned reads are mapped to reference genome by applying BWA. To avoid mapping bias, the GATK is used to realign reads near indels (insertions and deletions) and recalibrate base quality controls. Whole-genome single nucleotide variations (SNVs) and indels are detected by LoFreq*, Mutect2, VarScan2, and Pindel. Last, SNVs and indels are compared with in silico off-target sites using Cas-OFFinder.},
}
@article {pmid33471328,
year = {2021},
author = {Das, A and Ghana, P and Rudrappa, B and Gandhi, R and Tavva, VS and Mohanty, A},
title = {Genome Editing of Rice by CRISPR-Cas: End-to-End Pipeline for Crop Improvement.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {115-134},
pmid = {33471328},
issn = {1940-6029},
abstract = {CRISPR-Cas resonates a revolutionary genome editing technology applicable through a horizon spreading across microbial organism to higher plant and animal. This technology can be harnessed with ease to understand the basic genetics of a living system by altering sequence of individual genes and characterizing their functions. The precision of this technology is unparallel. It allows very precise and targeted base pair level edits in the genome. Here, in the current chapter, we have provided end-to-end process outline on how to generate genome edited plants in crops like rice to evaluate for agronomic traits associated with yield, disease resistance and abiotic stress tolerance, etc. Genome editing process includes designing of gene editing strategy, vector construction, plant transformation, molecular screening, and phenotyping under control environment conditions. Furthermore, its application for development of commercial crop product may require additional processes, including field trials in the target geography for evaluation of product efficacy. Evaluation of genome edited lines in controlled greenhouse/net house or open field condition requires few generations for outcrossing with wild-type parent to eliminate and/or reduce any potential pleiotropic effect in the edited genome which may arise during the process. The genome edited plant selected for advancement shall harbor the genome with only the intended changes, which can be analyzed by various molecular techniques, advanced sequencing methods, and genomic data analysis tools. CRISPR-Cas-based genome editing has opened a plethora of opportunities in agriculture as well as human health.},
}
@article {pmid33469138,
year = {2021},
author = {Fernandes, LGV and Hornsby, RL and Nascimento, ALTO and Nally, JE},
title = {Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1768},
pmid = {33469138},
issn = {2045-2322},
support = {2019/20302-8//Fapesp/ ; 2014/50981-0//Fapesp/ ; },
abstract = {Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.},
}
@article {pmid33468705,
year = {2021},
author = {Singh, A and Gaur, M and Sharma, V and Khanna, P and Bothra, A and Bhaduri, A and Mondal, AK and Dash, D and Singh, Y and Misra, R},
title = {Comparative Genomic Analysis of Mycobacteriaceae Reveals Horizontal Gene Transfer-Mediated Evolution of the CRISPR-Cas System in the Mycobacterium tuberculosis Complex.},
journal = {mSystems},
volume = {6},
number = {1},
pages = {},
pmid = {33468705},
issn = {2379-5077},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are conserved genetic elements in many prokaryotes, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Although knowledge of CRISPR locus variability has been utilized in M. tuberculosis strain genotyping, its evolutionary path in Mycobacteriaceae is not well understood. In this study, we have performed a comparative analysis of 141 mycobacterial genomes and identified the exclusive presence of the CRISPR-Cas type III-A system in M. tuberculosis complex (MTBC). Our global phylogenetic analysis of CRISPR repeats and Cas10 proteins offers evidence of horizontal gene transfer (HGT) of the CRISPR-Cas module in the last common ancestor of MTBC and Mycobacterium canettii from a Streptococcus-like environmental bacterium. Additionally, our results show that the variation of CRISPR-Cas organization in M. tuberculosis lineages, especially in the Beijing sublineage of lineage 2, is due to the transposition of insertion sequence IS6110 The direct repeat (DR) region of the CRISPR-Cas locus acts as a hot spot for IS6110 insertion. We show in M. tuberculosis H37Rv that the repeat at the 5' end of CRISPR1 of the forward strand is an atypical repeat made up partly of IS-terminal inverted repeat and partly CRISPR DR. By tracing an undetectable spacer sequence in the DR region, the two CRISPR loci could theoretically be joined to reconstruct the ancestral single CRISPR-Cas locus organization, as seen in M. canettii This study retracing the evolutionary events of HGT and IS6110-driven genomic deletions helps us to better understand the strain-specific variations in M. tuberculosis lineages.IMPORTANCE Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is M. tuberculosis, the leading cause of bacterial infection worldwide. Recent evidence on the functionality of the CRISPR-Cas system in M. tuberculosis has brought back focus on these conserved genetic elements, present in many prokaryotes. Our study advances understanding of mycobacterial CRISPR-Cas origin and its diversity among the different species. We provide phylogenetic evidence of acquisition of CRISPR-Cas type III-A in the last common ancestor shared between MTBC and M. canettii, by HGT-mediated events. The most likely source of HGT was an environmental Firmicutes bacterium. Genomic mapping of the CRISPR loci showed the IS6110 transposition-driven variations in M. tuberculosis strains. Thus, this study offers insights into events related to the evolution of CRISPR-Cas in M. tuberculosis lineages.},
}
@article {pmid33468685,
year = {2021},
author = {Xiao, H and Wyler, E and Milek, M and Grewe, B and Kirchner, P and Ekici, A and Silva, ABOV and Jungnickl, D and Full, F and Thomas, M and Landthaler, M and Ensser, A and Überla, K},
title = {CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs.},
journal = {mBio},
volume = {12},
number = {1},
pages = {},
pmid = {33468685},
issn = {2150-7511},
abstract = {The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern.IMPORTANCE To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.},
}
@article {pmid33454599,
year = {2021},
author = {Ashraf, MU and Salman, HM and Khalid, MF and Khan, MHF and Anwar, S and Afzal, S and Idrees, M and Chaudhary, SU},
title = {CRISPR-Cas13a mediated targeting of hepatitis C virus internal-ribosomal entry site (IRES) as an effective antiviral strategy.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {136},
number = {},
pages = {111239},
doi = {10.1016/j.biopha.2021.111239},
pmid = {33454599},
issn = {1950-6007},
abstract = {Hepatitis C is an inflammatory liver disease caused by the single-stranded RNA (ssRNA) hepatitis C virus (HCV). The genetic diversity of the virus and quasispecies produced during replication have resulted in viral resistance to direct-acting antivirals (DAAs) as well as impediments in vaccine development. The recent adaptation of CRISPR-Cas as an alternative antiviral approach has demonstrated degradation of viral nucleic acids in eukaryotes. In particular, the CRISPR-effector Cas13 enzyme has been shown to target ssRNA viruses effectively. In this work, we have employed Cas13a to knockdown HCV in mammalian cells. Using a computational screen, we identified several potential Cas13a target sites within highly conserved regions of the HCV internal ribosomal entry site (IRES). Our results demonstrate significant inhibition of HCV replication as well as translation in huh-7.5 cells with minimal effects on cell viability. These findings were validated using a multi-modality approach involving qRT-PCR, luciferase assay, and MTT cell viability assay. In conclusion, the CRISPR-Cas13a system efficiently targets HCV in vitro, suggesting its potential as a programmable therapeutic antiviral strategy.},
}
@article {pmid33453817,
year = {2020},
author = {Pinci, F and Gaidt, MM and Jung, C and Kuut, G and Jackson, MA and Bauernfried, S and Hornung, V},
title = {C-tag TNF: a reporter system to study TNF shedding.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {52},
pages = {18065-18075},
doi = {10.1074/jbc.RA120.015248},
pmid = {33453817},
issn = {1083-351X},
abstract = {TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.},
}
@article {pmid33461211,
year = {2021},
author = {Rostøl, JT and Xie, W and Kuryavyi, V and Maguin, P and Kao, K and Froom, R and Patel, DJ and Marraffini, LA},
title = {The Card1 nuclease provides defence during type-III CRISPR immunity.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-021-03206-x},
pmid = {33461211},
issn = {1476-4687},
abstract = {During the prokaryotic type III CRISPR-Cas immune response, infection triggers the production of cyclic oligoadenylates, which bind and activate CARF domain-containing proteins1,2. Many type III loci are associated with proteins in which the CARF domain is fused to an endonuclease-like domain3,4; however, with the exception of the well-characterized Csm6/Csx1 RNases5,6, whether and how these inducible effectors provide defense is not known. Here we investigated one of such type III CRISPR accessory proteins, Card1. Card1 forms a symmetrical dimer with a large central cavity between its CARF and restriction endonuclease (REase) domains that binds cA4. Ligand binding results in a conformational change where individual monomers rotate relative to each other to form a more compact dimeric scaffold wherein a Mn cation coordinates to the catalytic residues and activates the cleavage of single, but not double, stranded nucleic acids (DNA and RNA). In vivo, Card1 activation induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used by CRISPR systems to provide immunity.},
}
@article {pmid33349126,
year = {2021},
author = {Cornu, TI and Mussolino, C and Müller, MC and Wehr, C and Kern, WV and Cathomen, T},
title = {HIV Gene Therapy: An Update.},
journal = {Human gene therapy},
volume = {32},
number = {1-2},
pages = {52-65},
doi = {10.1089/hum.2020.159},
pmid = {33349126},
issn = {1557-7422},
abstract = {Progress in antiretroviral therapy has considerably reduced mortality and notably improved the quality of life of individuals infected with HIV since the pandemic began some 40 years ago. However, drug resistance, treatment-associated toxicity, adherence to medication, and the need for lifelong therapy have remained major challenges. While the development of an HIV vaccine has remained elusive, considerable progress in developing innovative cell and gene therapies to treat HIV infection has been made. This includes immune cell therapies, such as chimeric antigen receptor T cells to target HIV infected cells, as well as gene therapies and genome editing strategies to render the patient's immune system resistant to HIV. Nonetheless, all of these attempts to achieve a functional cure in HIV patients have failed thus far. This review introduces the clinical as well as the technical challenges of treating HIV infection, and summarizes the most promising cell and gene therapy concepts that have aspired to bring about functional cure for people living with HIV. It further discusses socioeconomic aspects as well as future directions for developing cell and gene therapies with a potential to be an effective one-time treatment with minimal toxicity.},
}
@article {pmid33243861,
year = {2020},
author = {Jin, X and Simmons, SK and Guo, A and Shetty, AS and Ko, M and Nguyen, L and Jokhi, V and Robinson, E and Oyler, P and Curry, N and Deangeli, G and Lodato, S and Levin, JZ and Regev, A and Zhang, F and Arlotta, P},
title = {In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6520},
pages = {},
doi = {10.1126/science.aaz6063},
pmid = {33243861},
issn = {1095-9203},
support = {U01 MH115727/MH/NIMH NIH HHS/United States ; R01 MH096066/MH/NIMH NIH HHS/United States ; P50 MH094271/MH/NIMH NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; //Howard Hughes Medical Insititue/International ; },
mesh = {Animals ; Ankyrins/genetics/metabolism ; Autistic Disorder/*genetics/*pathology ; Brain/*abnormalities ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Frameshift Mutation ; Gene Expression Profiling ; Genetic Loci ; Humans ; Mice ; Neuroglia/metabolism/*pathology ; Neurons/metabolism/*pathology ; Repressor Proteins/genetics ; Risk ; Transcription Factors/genetics ; },
abstract = {The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA-sequencing of perturbed cells in the postnatal brain. We identified cell type-specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.},
}
@article {pmid33220342,
year = {2021},
author = {Katsuma, S and Shoji, K and Suzuki, Y and Kiuchi, T},
title = {CRISPR/Cas9-mediated mutagenesis of Ago2 and Siwi in silkworm cultured cells.},
journal = {Gene},
volume = {768},
number = {},
pages = {145314},
doi = {10.1016/j.gene.2020.145314},
pmid = {33220342},
issn = {1879-0038},
mesh = {Animals ; Argonaute Proteins/*genetics ; Bombyx/*cytology/genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Gene Editing ; Insect Proteins/genetics ; Loss of Function Mutation ; Mutagenesis, Site-Directed/*methods ; RNA, Small Interfering/genetics ; RNA, Viral/genetics ; Signal Transduction ; Tymoviridae/*genetics ; },
abstract = {The BmN-4 cell line, originated from the silkworm Bombyx mori ovary, possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. We performed CRISPR/Cas9-mediated genome editing of Ago2 and Siwi, which are the core factors for siRNA and piRNA pathways, respectively, to understand the importance of the two distinct small RNA pathways in this cell line. We found that approximately half of the alleles contained loss-of-function mutations in both Ago2- and Siwi-mutated cells. The mutated cells grew at a slower rate compared to the control cells, strongly suggesting that the siRNA and piRNA pathways are both crucial for the normal growth of BmN-4 cells. The amounts of piRNAs decreased markedly in the Siwi-mutated cells, but global de-repression of transposable elements was not observed. Although the RNA amount of latently infected RNA virus, Bombyx mori macula-like virus (BmLV), increased in both Ago2- and Siwi-mutated cells, the siRNA and piRNA pathways showed a bias toward targeting BmLV genomic and subgenomic RNA, respectively. These results indicate the common, specific, and crucial roles of the two small RNA pathways in B. mori cultured cells.},
}
@article {pmid33172989,
year = {2020},
author = {Zhang, H and Zoued, A and Liu, X and Sit, B and Waldor, MK},
title = {Type I interferon remodels lysosome function and modifies intestinal epithelial defense.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {47},
pages = {29862-29871},
pmid = {33172989},
issn = {1091-6490},
support = {/HHMI/Howard Hughes Medical Institute/United States ; R01 AI042347/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Epithelial Cells/chemistry/cytology/*immunology/metabolism ; Gene Expression Regulation, Bacterial/immunology ; HT29 Cells ; Host-Pathogen Interactions/genetics/immunology ; Humans ; Hydrogen-Ion Concentration ; Immunity, Innate ; Interferon Type I/*metabolism ; Intestinal Mucosa/cytology/*immunology/microbiology ; Lysosomes/chemistry/immunology/*metabolism ; Mice ; Mice, Knockout ; Necroptosis/immunology ; Peptide Hydrolases/metabolism ; Proteomics ; Receptor, Interferon alpha-beta/genetics/metabolism ; Salmonella Infections/*immunology/microbiology ; Salmonella typhimurium/immunology/pathogenicity ; Signal Transduction/immunology ; Virulence/immunology ; Virulence Factors/genetics/metabolism ; },
abstract = {Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I IFN (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity, and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including IFITM3, SLC15A3, and CNP contribute to lysosome acidification. IFN-I-dependent lysosome acidification was associated with elevated intracellular Salmonella virulence gene expression, rupture of the Salmonella-containing vacuole, and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium where Salmonella initiates infection, indicating that IFN-I signaling can modify innate defense in the epithelial compartment. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.},
}
@article {pmid33168709,
year = {2020},
author = {Yu, X and Zhao, Q and Li, X and Chen, Y and Tian, Y and Liu, S and Xiong, W and Huang, P},
title = {Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {47},
pages = {29894-29903},
pmid = {33168709},
issn = {1091-6490},
mesh = {Animals ; COS Cells ; CRISPR-Cas Systems/genetics ; Chlorocebus aethiops ; Deafness/*genetics/pathology ; Disease Models, Animal ; Gene Knock-In Techniques ; HEK293 Cells ; Hair Cells, Auditory, Inner/metabolism/*pathology ; Humans ; Mechanotransduction, Cellular/*genetics ; Membrane Proteins/*genetics/isolation & purification/*metabolism ; Mice ; Mice, Transgenic ; Point Mutation ; Protein Binding/genetics ; Two-Hybrid System Techniques ; },
abstract = {Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells.},
}
@article {pmid32887745,
year = {2020},
author = {Llamosas, N and Arora, V and Vij, R and Kilinc, M and Bijoch, L and Rojas, C and Reich, A and Sridharan, B and Willems, E and Piper, DR and Scampavia, L and Spicer, TP and Miller, CA and Holder, JL and Rumbaugh, G},
title = {SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {40},
number = {41},
pages = {7980-7994},
pmid = {32887745},
issn = {1529-2401},
support = {R01 MH096847/MH/NIMH NIH HHS/United States ; R01 MH113648/MH/NIMH NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Size ; Cells, Cultured ; Dendrites/*physiology ; Excitatory Postsynaptic Potentials/genetics ; Female ; Gene Deletion ; Humans ; Nerve Net/*physiology ; Nervous System/*growth & development ; Neurodevelopmental Disorders/genetics ; Pluripotent Stem Cells ; Synapses/*physiology ; ras GTPase-Activating Proteins/*genetics/*physiology ; },
abstract = {SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.},
}
@article {pmid32745561,
year = {2020},
author = {Yan, Y and Ziemek, J and Schetelig, MF},
title = {CRISPR/Cas9 mediated disruption of the white gene leads to pigmentation deficiency and copulation failure in Drosophila suzukii.},
journal = {Journal of insect physiology},
volume = {126},
number = {},
pages = {104091},
doi = {10.1016/j.jinsphys.2020.104091},
pmid = {32745561},
issn = {1879-1611},
mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Copulation ; Drosophila/*genetics/physiology ; Drosophila Proteins/*genetics ; Eye Proteins/*genetics ; Female ; Genes, Insect ; Insect Control/methods ; Male ; Mutation ; Pigmentation/*genetics ; *Sexual Behavior, Animal ; },
abstract = {The Spotted-wing Drosophila (Drosophila suzukii) is a devastating invasive pest of fruit crops. In D. melanogaster, the white (w) gene was associated with pigmentation and mating behavior, which are also important aspects to understand the invasion biology as well as to develop control strategies for D. suzukii. Here, we show that the generation of D. suzukii white-eyed mutants by CRISPR/Cas9 mutagenesis of the w gene resulted in the complete failure of copulation when w- males were individually paired with w- females in small circular arenas (diameter 0.7 cm) for 24 h. Further analysis showed that the mating defect was associated with w- males and could not be rectified by two years of inbreeding by crossing sibling w- females with w+ males, dim red illumination, male-female sexual training, changing to large arenas (diameter 3.5 cm), or different sex ratios. Profound pigmentation deficiency was detected in the compound eyes, ocelli, Malpighian tubules and testis sheaths in the w- flies. Specifically, testis imaging showed that w- males failed to deposit any pigments into pigment cells of the testis sheath, and produced smaller sperms and less seminal fluid compared to those from wildtype males. Together these observations suggest that the w gene plays an essential role in the regulation of sexual behavior and reproduction in D. suzukii. The similarities and differences in w gene function between D. suzukii and D. melanogaster in the context of pigmentation and mating behavior are discussed.},
}
@article {pmid32518161,
year = {2020},
author = {Hu, L and Li, H and Chi, Z and He, J},
title = {Loss of the RNA-binding protein Rbm15 disrupts liver maturation in zebrafish.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {33},
pages = {11466-11472},
pmid = {32518161},
issn = {1083-351X},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; *Gene Deletion ; *Gene Expression Regulation, Developmental ; Hepatocytes/cytology/metabolism ; Liver/cytology/*embryology ; Zebrafish/*embryology/genetics ; },
abstract = {Liver organogenesis begins with hepatic precursors in the foregut endoderm, followed by hepatoblast specification, differentiation, outgrowth, and maturation for the formation of functional hepatocytes. Although several signaling pathways and critical factors that regulate liver specification, differentiation, and proliferation have been identified, little is known about how liver maturation is regulated. Here, we used a screen for mutations affecting liver development in zebrafish and identified a cq96 mutant that exhibits a specific defect in liver maturation. Results from positional cloning revealed that cq96 encodes an RNA-binding protein, Rbm15, which is an evolutionarily conserved Spen family protein and known to play a crucial role in RNA m6A modification, nuclear export, and alternative splicing. However, a function of Rbm15 in embryonic liver development has not been reported. We found that Rbm15 is specifically expressed in the liver after its differentiation. CRISPR/Cas9-mediated loss of rbm15 repressed hepatic maturation, but did not affect hepatoblast specification, differentiation, and hepatocyte proliferation and apoptosis. Additional experiments disclosed that the mTOR complex 1 (mTORC1) pathway is highly activated in rbm15-deficient hepatocytes. Moreover, rapamycin treatment partially restored normal hepatic gene expression as well as the nuclear location of the transcription factor Hnf4a. Taken together, these results reveal an unexpected role of Rbm15 in liver maturation.},
}
@article {pmid32349563,
year = {2020},
author = {Tromp, TR and Stroes, ESG and Hovingh, GK},
title = {Gene-based therapy in lipid management: the winding road from promise to practice.},
journal = {Expert opinion on investigational drugs},
volume = {29},
number = {5},
pages = {483-493},
doi = {10.1080/13543784.2020.1757070},
pmid = {32349563},
issn = {1744-7658},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cardiovascular Diseases/etiology/*prevention & control ; Genetic Therapy/*methods ; Heart Disease Risk Factors ; Humans ; Hypercholesterolemia/complications/genetics/*therapy ; Lipid Metabolism/genetics ; Oligonucleotides, Antisense/administration & dosage ; },
abstract = {INTRODUCTION: Cardiovascular disease (CVD) is a leading cause of morbidity and mortality. High plasma low-density lipoprotein cholesterol (LDL-C) levels are a key CVD-risk factor. Triglyceride-rich remnant particles and lipoprotein(a) (Lp[a]) are also causally related to CVD. Consequently, therapeutic strategies for lowering LDL-C and triglyceride levels are widely used in routine clinical practice; however, specific Lp(a) lowering agents are not available. Many patients do not achieve guideline-recommended lipid levels with currently available therapies; hence, novel targets and treatment modalities are eagerly sought.<br><br>AREAS COVERED: We discuss the milestones on the trajectory toward the full application of gene-based therapies in daily clinical practice. We describe the different methods, ranging from antisense oligonucleotides to liver-directed gene therapy and Crispr-cas9 modification to target the pivotal players in lipid metabolism: PCSK9, APOB, ANGPTL3, Lp(a), LDLR, and apoC-III.<br><br>EXPERT OPINION: While acknowledging their different stages of development, gene-based therapies are likely to invoke a paradigm shift in lipid management because they allow us to target previously undruggable targets. Moreover, their low dosing frequency, high target selectivity, and relatively predictable adverse event profile are considered major advantages over current lipid-lowering therapies.},
}
@article {pmid32307772,
year = {2020},
author = {Chiu, YW and Hori, Y and Ebinuma, I and Sato, H and Hara, N and Ikeuchi, T and Tomita, T},
title = {Identification of calcium and integrin-binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9-based screening system.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {6},
pages = {7661-7674},
doi = {10.1096/fj.201902966RR},
pmid = {32307772},
issn = {1530-6860},
mesh = {Alzheimer Disease/metabolism ; Amyloid Precursor Protein Secretases/metabolism ; Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/metabolism ; Animals ; Brain/metabolism ; CRISPR-Cas Systems/*physiology ; Calcium-Binding Proteins/*metabolism ; Carrier Proteins/metabolism ; Cell Line ; Cell Line, Tumor ; Cell Membrane/metabolism ; HEK293 Cells ; Humans ; Mice ; Neurons/metabolism ; Protein Binding/physiology ; Protein Transport/physiology ; Synapsins/metabolism ; Up-Regulation/physiology ; },
abstract = {The aberrant metabolism of amyloid β peptide (Aβ) has been implicated in the etiology of Alzheimer disease (AD). Aβ is produced via the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. However, the precise regulatory mechanism of Aβ generation still remains unclear. To gain a better understanding of the molecular mechanism of Aβ production, we established a genetic screening method based on the CRISPR/Cas9 system to identify novel regulators of Aβ production. We successfully identified calcium and integrin-binding protein 1 (CIB1) as a potential negative regulator of Aβ production. The disruption of Cib1 significantly upregulated Aβ levels. In addition, immunoprecipitation experiments demonstrated that CIB1 interacts with the γ-secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell-surface localization of mature Nicastrin (Nct), which is a component of the γ-secretase complex, without changing the intrinsic activity of γ-secretase. Finally, we confirmed using the single-cell RNA-seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aβ production via controlling the subcellular localization of γ-secretase, suggesting CIB1 is involved in the development of AD.},
}
@article {pmid32302524,
year = {2020},
author = {Tian, S and Liu, Y and Wu, H and Liu, H and Zeng, J and Choi, MY and Chen, H and Gerhard, R and Dong, M},
title = {Genome-Wide CRISPR Screen Identifies Semaphorin 6A and 6B as Receptors for Paeniclostridium sordellii Toxin TcsL.},
journal = {Cell host &amp; microbe},
volume = {27},
number = {5},
pages = {782-792.e7},
pmid = {32302524},
issn = {1934-6069},
support = {R21 NS106159/NS/NINDS NIH HHS/United States ; R01 NS080833/NS/NINDS NIH HHS/United States ; R01 HL146134/HL/NHLBI NIH HHS/United States ; R01 HL093242/HL/NHLBI NIH HHS/United States ; R01 AI132387/AI/NIAID NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; R01 HL130845/HL/NHLBI NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; },
mesh = {A549 Cells ; Animals ; Bacterial Proteins ; Bacterial Toxins/*metabolism ; Binding Sites ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clostridium sordellii/*genetics/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endothelial Cells/metabolism ; Female ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Lung Neoplasms ; Male ; Mice ; Protein Binding ; Semaphorins/*genetics/*isolation &amp; purification/metabolism ; Virulence Factors/metabolism ; },
abstract = {The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.},
}
@article {pmid32277852,
year = {2020},
author = {Przanowska, RK and Sobierajska, E and Su, Z and Jensen, K and Przanowski, P and Nagdas, S and Kashatus, JA and Kashatus, DF and Bhatnagar, S and Lukens, JR and Dutta, A},
title = {miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {6},
pages = {7687-7702},
pmid = {32277852},
issn = {1530-6860},
support = {18PRE33990261/AHA/American Heart Association-American Stroke Association/United States ; R01 AR067712/AR/NIAMS NIH HHS/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; T32 GM007267/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Cell Proliferation/genetics ; HEK293 Cells ; Humans ; Mice ; Mice, Knockout ; MicroRNAs/*genetics ; Mitochondria/*genetics ; Muscle Development/*genetics ; Muscle, Skeletal/*physiology ; Muscular Diseases/genetics ; Myoblasts, Skeletal/*physiology ; },
abstract = {miR-206, miR-1a-1, and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this miRNA family is hypothesized to play an essential role in differentiation, a triple knock-out (tKO) of the three genes has not been done to test this hypothesis. We report that tKO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected derepression of the miRNA targets. Surprisingly, their mitochondrial function is diminished. tKO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two tKO mice survive and grow normally to adulthood with smaller myofiber diameter, diminished physical performance, and an increase in PAX7 positive satellite cells. Thus, unlike other miRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.},
}
@article {pmid32213923,
year = {2020},
author = {Lim, KRQ and Nguyen, Q and Dzierlega, K and Huang, Y and Yokota, T},
title = {CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy.},
journal = {Genes},
volume = {11},
number = {3},
pages = {},
pmid = {32213923},
issn = {2073-4425},
support = {FDN 143251//CIHR/Canada ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Gene Editing/methods ; Haplorhini ; Murinae ; Muscular Dystrophy, Duchenne/*genetics/pathology ; Rabbits ; Swine ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.},
}
@article {pmid32164255,
year = {2020},
author = {Lanigan, TM and Kopera, HC and Saunders, TL},
title = {Principles of Genetic Engineering.},
journal = {Genes},
volume = {11},
number = {3},
pages = {},
pmid = {32164255},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Targeting/methods ; Gene Transfer Techniques ; Genetic Engineering/*methods/standards/trends ; Humans ; },
abstract = {Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.},
}
@article {pmid32100378,
year = {2020},
author = {Kim, GD and Lee, JH and Song, S and Kim, SW and Han, JS and Shin, SP and Park, BC and Park, TS},
title = {Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {4},
pages = {5688-5696},
doi = {10.1096/fj.201903035R},
pmid = {32100378},
issn = {1530-6860},
mesh = {Animals ; Animals, Genetically Modified/genetics/growth &amp; development/*metabolism ; *CRISPR-Cas Systems ; Chickens ; *Gene Editing ; Germ Cells/cytology/*metabolism ; Muscle, Skeletal/cytology/*metabolism ; Myostatin/antagonists &amp; inhibitors/*physiology ; Phenotype ; },
abstract = {Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off-target effects, including the cleavage of unintended targets, limits the practical applications of Cas9-mediated genome editing. In this study, to minimize the off-target effects of this technology, we utilized D10A-Cas9 nickase to generate myostatin-knockout (MSTN KO) chickens via primordial germ cells. D10A-Cas9 nickase (Cas9n)-mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild-type chickens. Our results demonstrate that the D10A-Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome-edited chickens in practical industrialization and as animal models of human diseases.},
}
@article {pmid31999448,
year = {2020},
author = {Siemon, T and Wang, Z and Bian, G and Seitz, T and Ye, Z and Lu, Y and Cheng, S and Ding, Y and Huang, Y and Deng, Z and Liu, T and Christmann, M},
title = {Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {6},
pages = {2760-2765},
doi = {10.1021/jacs.9b12940},
pmid = {31999448},
issn = {1520-5126},
mesh = {CRISPR-Cas Systems ; Escherichia coli/genetics ; *Metabolic Engineering ; Plants/*chemistry ; Saccharomyces cerevisiae/genetics ; Sesquiterpenes, Guaiane/chemical synthesis/*chemistry ; },
abstract = {Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.},
}
@article {pmid31931564,
year = {2020},
author = {Alyami, MZ and Alsaiari, SK and Li, Y and Qutub, SS and Aleisa, FA and Sougrat, R and Merzaban, JS and Khashab, NM},
title = {Cell-Type-Specific CRISPR/Cas9 Delivery by Biomimetic Metal Organic Frameworks.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {4},
pages = {1715-1720},
doi = {10.1021/jacs.9b11638},
pmid = {31931564},
issn = {1520-5126},
mesh = {Animals ; *Biomimetics ; *CRISPR-Cas Systems ; HeLa Cells ; Heterografts ; Humans ; MCF-7 Cells ; Metal-Organic Frameworks/*chemistry ; Mice ; },
abstract = {Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer cell coated zeolitic imidazolate frameworks (ZIFs) for targeted and cell-specific delivery of this genome editing machinery. Coating ZIF-8 that is encapsulating CRISPR/Cas9 (CC-ZIF) with a cancer cell membrane resulted in the uniformly covered C3-ZIF(cell membrane type). Incubation of C3-ZIFMCF with MCF-7, HeLa, HDFn, and aTC cell lines showed the highest uptake by MCF-7 cells and negligible uptake by the healthy cells (i.e., HDFn and aTC). As to genome editing, a 3-fold repression in the EGFP expression was observed when MCF-7 were transfected with C3-ZIFMCF compared to 1-fold repression in the EGFP expression when MCF-7 were transfected with C3-ZIFHELA. In vivo testing confirmed the selectivity of C3-ZIFMCF to accumulate in MCF-7 tumor cells. This supports the ability of this biomimetic approach to match the needs of cell-specific targeting, which is unquestionably the most critical step in the future translation of genome editing technologies.},
}
@article {pmid31893458,
year = {2020},
author = {Bao, A and Tran, LP and Cao, D},
title = {CRISPR/Cas9-Based Gene Editing in Soybean.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2107},
number = {},
pages = {349-364},
doi = {10.1007/978-1-0716-0235-5_19},
pmid = {31893458},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Cotyledon/genetics/*growth &amp; development ; Gene Editing/*methods ; Mutation ; Plant Breeding ; Soybeans/genetics/*growth &amp; development ; Tissue Culture Techniques ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean, a number of laboratories have obtained mutants by CRISPR/Cas9 system; however, there has been not yet a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single guide RNA) transgenes.},
}
@article {pmid31882399,
year = {2020},
author = {Ianiri, G and Fang, YF and Dahlmann, TA and Clancey, SA and Janbon, G and Kück, U and Heitman, J},
title = {Mating-Type-Specific Ribosomal Proteins Control Aspects of Sexual Reproduction in Cryptococcus neoformans.},
journal = {Genetics},
volume = {214},
number = {3},
pages = {635-649},
pmid = {31882399},
issn = {1943-2631},
support = {R01 AI039115/AI/NIAID NIH HHS/United States ; R01 AI050113/AI/NIAID NIH HHS/United States ; R37 AI039115/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Cryptococcus neoformans/*genetics/growth &amp; development ; Fungal Proteins/genetics ; Genes, Mating Type, Fungal/*genetics ; Haploidy ; Phenotype ; Reproduction/*genetics ; Ribosomal Proteins/*genetics ; },
abstract = {The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, and RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and RPL39 Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22αΔ or RPL22α/rpl22aΔ mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology, the RPL22 alleles were exchanged in haploid MATα and MATa strains of C. neoformans These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidating functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.},
}
@article {pmid33453283,
year = {2021},
author = {Koay, TW and Osterhof, C and Orlando, IMC and Keppner, A and Andre, D and Yousefian, S and Alonso, MS and Correia, M and Markworth, R and Schödel, J and Hankeln, T and Hoogewijs, D},
title = {Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {100291},
doi = {10.1016/j.jbc.2021.100291},
pmid = {33453283},
issn = {1083-351X},
abstract = {Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell type-specific expression, regulation and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that -in addition to testes- ADGB is prominently expressed in the female reproductive tract, lungs and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested co-regulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.},
}
@article {pmid33452819,
year = {2021},
author = {Barbour, A and Glogauer, J and Grinfeld, L and Ostadsharif Memar, R and Fine, N and Tenenbaum, H and Glogauer, M},
title = {The role of CRISPR-Cas in advancing precision periodontics.},
journal = {Journal of periodontal research},
volume = {},
number = {},
pages = {},
doi = {10.1111/jre.12846},
pmid = {33452819},
issn = {1600-0765},
abstract = {The significant advancement of molecular biology has revolutionized medicine and provided important technologies to further clinical research development. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are DNA sequences derived from bacteriophages which have previously infected the bacterial species. The CRISPR-Cas system plays a key role in bacterial defense by detecting and destroying DNA fragments during subsequent bacteriophage invasions. The Cas9 enzyme recognizes and cleaves new invading CRISPR-complementary DNA sequences. Researchers have taken advantage of this biological device to manipulate microbes' genes and develop novel therapeutics to tackle systemic disease. In this review, we discuss the potential of utilizing CRISPR-Cas systems in the periodontal field to develop personalized periodontal care. We summarize promising attempts to bring this technology to the clinical setting. Finally, we provide insights regarding future developments to best utilize the CRISPR-Cas systems to advance precision periodontics. Although further research is imperative to evaluate the safety and potential of using CRISPR-Cas to develop precision periodontics approaches, few studies showed promising data to support the investment into this important technology in the dental sector. CRISPR-Cas9 can be a useful tool to create knockouts in vitro and in vivo as a screening tool to identify cellular pathways involved in the pathogenesis of periodontitis. Alternative CRISPR systems such as CRISPRa, CRISPRi, and Cas13 can be used to modify the transcriptome and gene expression of genes involved in periodontitis progression. CRISPR systems such as Cas3 can be used to target the periodontal biofilm and to develop new strategies to reduce or eliminate periodontal pathogens. Currently, the utility of CRISPR-Cas applications in clinical settings is limited. Through this review, we hope to foster further discussion in the periodontal research and clinical communities with respect to the potential clinical application of novel, CRISPR-Cas based, therapeutics for periodontitis.},
}
@article {pmid33449167,
year = {2021},
author = {Brandes, RP and Dueck, A and Engelhardt, S and Kaulich, M and Kupatt, C and De Angelis, MT and Leisegang, MS and le Noble, F and Moretti, A and Müller, OJ and Skryabin, BV and Thum, T and Wurst, W},
title = {DGK and DZHK position paper on genome editing: basic science applications and future perspective.},
journal = {Basic research in cardiology},
volume = {116},
number = {1},
pages = {2},
pmid = {33449167},
issn = {1435-1803},
abstract = {For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.},
}
@article {pmid33338421,
year = {2021},
author = {Hoffmann, HH and Schneider, WM and Rozen-Gagnon, K and Miles, LA and Schuster, F and Razooky, B and Jacobson, E and Wu, X and Yi, S and Rudin, CM and MacDonald, MR and McMullan, LK and Poirier, JT and Rice, CM},
title = {TMEM41B Is a Pan-flavivirus Host Factor.},
journal = {Cell},
volume = {184},
number = {1},
pages = {133-148.e20},
doi = {10.1016/j.cell.2020.12.005},
pmid = {33338421},
issn = {1097-4172},
support = {R01 AI124690/AI/NIAID NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Asian Continental Ancestry Group/genetics ; Autophagy ; COVID-19/genetics/metabolism/virology ; CRISPR-Cas Systems ; Cell Line ; Flavivirus/*physiology ; Flavivirus Infections/*genetics/immunology/metabolism/virology ; Gene Knockout Techniques ; Genome-Wide Association Study ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Membrane Proteins/genetics/*metabolism ; Polymorphism, Single Nucleotide ; SARS-CoV-2/physiology ; Virus Replication ; Yellow fever virus/physiology ; Zika Virus/physiology ; },
abstract = {Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.},
}
@article {pmid33139551,
year = {2020},
author = {Takao, T and Sato, M and Maruyama, T},
title = {Optogenetic regulation of embryo implantation in mice using photoactivatable CRISPR-Cas9.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {46},
pages = {28579-28581},
pmid = {33139551},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Embryo Implantation ; Fertility ; Leukemia Inhibitory Factor/genetics/*metabolism ; Mice, Inbred ICR ; *Optogenetics ; },
abstract = {Embryo implantation is achieved upon successful interaction between a fertilized egg and receptive endometrium and is mediated by spatiotemporal expression of implantation-associated molecules including leukemia inhibitory factor (LIF). Here we demonstrate, in mice, that LIF knockdown via a photoactivatable CRISPR-Cas9 gene editing system and illumination with a light-emitting diode can spatiotemporally disrupt fertility. This system enables dissection of spatiotemporal molecular mechanisms associated with embryo implantation and provides a therapeutic strategy for temporal control of reproductive functions in vivo.},
}
@article {pmid33075436,
year = {2020},
author = {Murphy, ZC and Getman, MR and Myers, JA and Burgos Villar, KN and Leshen, E and Kurita, R and Nakamura, Y and Steiner, LA},
title = {Codanin-1 mutations engineered in human erythroid cells demonstrate role of CDAN1 in terminal erythroid maturation.},
journal = {Experimental hematology},
volume = {91},
number = {},
pages = {32-38.e6},
pmid = {33075436},
issn = {1873-2399},
support = {R01 DK104920/DK/NIDDK NIH HHS/United States ; },
mesh = {Acetylation ; Anemia, Dyserythropoietic, Congenital/blood/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cell Nucleus/ultrastructure ; Cell Survival ; Chromatin/ultrastructure ; Erythroid Cells/*cytology/metabolism ; Erythropoiesis/*genetics/physiology ; Exons/genetics ; Gene Editing ; Glycoproteins/deficiency/*genetics/physiology ; Histone Code ; Humans ; Nuclear Proteins/deficiency/*genetics/physiology ; Phenotype ; Protein Processing, Post-Translational ; },
abstract = {The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology.},
}
@article {pmid33020616,
year = {2020},
author = {Matharu, N and Ahituv, N},
title = {Modulating gene regulation to treat genetic disorders.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {11},
pages = {757-775},
doi = {10.1038/s41573-020-0083-7},
pmid = {33020616},
issn = {1474-1784},
mesh = {Animals ; CRISPR-Cas Systems/drug effects/genetics ; Gene Expression Regulation/*drug effects/genetics ; Genetic Diseases, Inborn/*drug therapy/genetics ; Humans ; Mutation/drug effects/genetics ; Pharmaceutical Preparations/*administration &amp; dosage ; },
abstract = {Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements - promoters, enhancers, silencers and insulators, subsequently changing their target gene expression levels to achieve therapeutic benefits - an approach termed cis-regulation therapy (CRT). Here, we review emerging CRT technologies and assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed.},
}
@article {pmid33020605,
year = {2020},
author = {Leech, R and Sampath, K},
title = {A CRISPR cut for messenger RNAs.},
journal = {Lab animal},
volume = {49},
number = {11},
pages = {317-319},
doi = {10.1038/s41684-020-00661-3},
pmid = {33020605},
issn = {1548-4475},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; RNA, Messenger/genetics ; },
}
@article {pmid32601291,
year = {2020},
author = {Kamble, PG and Hetty, S and Vranic, M and Almby, K and Castillejo-López, C and Abalo, XM and Pereira, MJ and Eriksson, JW},
title = {Proof-of-concept for CRISPR/Cas9 gene editing in human preadipocytes: Deletion of FKBP5 and PPARG and effects on adipocyte differentiation and metabolism.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10565},
pmid = {32601291},
issn = {2045-2322},
mesh = {Adipocytes/*metabolism ; Adipogenesis/genetics ; Adipose Tissue/metabolism ; Adult ; Aged ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Humans ; Middle Aged ; PPAR gamma/genetics ; Proof of Concept Study ; Tacrolimus Binding Proteins/genetics ; },
abstract = {CRISPR/Cas9 has revolutionized the genome-editing field. So far, successful application in human adipose tissue has not been convincingly shown. We present a method for gene knockout using electroporation in preadipocytes from human adipose tissue that achieved at least 90% efficiency without any need for selection of edited cells or clonal isolation. We knocked out the FKBP5 and PPARG genes in preadipocytes and studied the resulting phenotypes. PPARG knockout prevented differentiation into adipocytes. Conversely, deletion of FKBP51, the protein coded by the FKBP5 gene, did not affect adipogenesis. Instead, it markedly modulated glucocorticoid effects on adipocyte glucose metabolism and, furthermore, we show some evidence of altered transcriptional activity of glucocorticoid receptors. This has potential implications for the development of insulin resistance and type 2 diabetes. The reported method is simple, easy to adapt, and enables the use of human primary preadipocytes instead of animal adipose cell models to assess the role of key genes and their products in adipose tissue development, metabolism and pathobiology.},
}
@article {pmid32576837,
year = {2020},
author = {Lamsfus-Calle, A and Daniel-Moreno, A and Antony, JS and Epting, T and Heumos, L and Baskaran, P and Admard, J and Casadei, N and Latifi, N and Siegmund, DM and Kormann, MSD and Handgretinger, R and Mezger, M},
title = {Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10133},
pmid = {32576837},
issn = {2045-2322},
mesh = {Anemia, Sickle Cell/*genetics/therapy ; Antigens, CD34 ; *CRISPR-Cas Systems ; Cells, Cultured ; Fetal Hemoglobin/*genetics ; Gene Editing/*methods ; Gene Expression/genetics ; Humans ; Kruppel-Like Transcription Factors/*genetics ; Molecular Targeted Therapy ; Mutation ; Repressor Proteins/*genetics ; gamma-Globins/*genetics ; },
abstract = {β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.},
}
@article {pmid32527526,
year = {2020},
author = {Yang, Y and Liu, G and Chen, X and Liu, M and Zhan, C and Liu, X and Bai, Z},
title = {High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in Pichia pastoris.},
journal = {Enzyme and microbial technology},
volume = {138},
number = {},
pages = {109556},
doi = {10.1016/j.enzmictec.2020.109556},
pmid = {32527526},
issn = {1879-0909},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; Genome, Fungal/genetics ; Nucleotide Motifs ; Plasmids/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/chemistry/*genetics ; Saccharomycetales/*genetics/growth &amp; development ; },
abstract = {Pichia pastoris is a methylotrophic yeast in which host heterologous expression of proteins has been developed owing to the strong inducible alcohol oxidase promoter (PAOX1). However, it is difficult to manipulate the genome in P. pastoris. Based on previous attempts to apply the CRISPR/Cas9 system in P. pastoris, a CRISPR/Cas9 system with episomal sgRNA plasmid was developed and 100 % genome editing efficiency, high multicopy gene editing and stable multigene editing were obtained without a sharp decline caused by multi-sgRNA. And 28/34 (∼82 %) sgRNAs tested were effective. The CGG may have a slightly higher and more stable cleavage efficiency than the other three NGG motifs, and a low GC content may be preferable for higher cleavage efficiency. This provides researchers with a stable genome editing tool that shows a high editing efficiency, shortening the experimentation period. Furthermore, we introduced dCas9 into P. pastoris and achieved target gene interference, expanding the CRISPR/Cas9 toolbox in P. pastoris.},
}
@article {pmid32321775,
year = {2020},
author = {Kagoya, Y and Guo, T and Yeung, B and Saso, K and Anczurowski, M and Wang, CH and Murata, K and Sugata, K and Saijo, H and Matsunaga, Y and Ohashi, Y and Butler, MO and Hirano, N},
title = {Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.},
journal = {Cancer immunology research},
volume = {8},
number = {7},
pages = {926-936},
doi = {10.1158/2326-6066.CIR-18-0508},
pmid = {32321775},
issn = {2326-6074},
mesh = {Allografts ; Animals ; Antigens, CD19/immunology ; CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Histocompatibility Antigens Class I/*chemistry/genetics ; Histocompatibility Antigens Class II/*chemistry/genetics ; Humans ; Immunotherapy, Adoptive/*methods ; Leukocytes, Mononuclear ; Lymphocyte Activation ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasms/immunology/metabolism/*therapy ; Receptors, Antigen, T-Cell/antagonists &amp; inhibitors/genetics/*immunology ; Receptors, Chimeric Antigen/*immunology ; },
abstract = {Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.},
}
@article {pmid33445135,
year = {2021},
author = {Zuo, F and Marcotte, H},
title = {Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing.},
journal = {Current opinion in biotechnology},
volume = {70},
number = {},
pages = {75-82},
doi = {10.1016/j.copbio.2020.12.015},
pmid = {33445135},
issn = {1879-0429},
abstract = {Typical traditional probiotics lactobacilli and bifidobacteria are gaining great interest to be developed as living diagnostics and therapeutics for improving human health. However, the mechanistic basis underlying their inherent health beneficial property remain incompletely understood which can slow down the translational pipeline in the functional food and pharmaceutical field. Efficient genome editing will advance the understanding of the molecular mechanism of the probiotics' physiological properties and their interaction with the host and the host microbiota, thereby further promote the development of next-generation designer probiotics with improved robustness and tailored functionalities. With the expansion of genome editing strategies such as CRISPR-Cas-based tools and IPSD assisted genome engineering as well as other synthetic biology technologies, the research and application of these health-promoting bacteria for the food and pharmaceutical industry will be further enhanced.},
}
@article {pmid33444542,
year = {2020},
author = {Li, Y and Bondy-Denomy, J},
title = {Anti-CRISPRs go viral: the infection biology of CRISPR-Cas inhibitors.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2020.12.007},
pmid = {33444542},
issn = {1934-6069},
abstract = {Bacteriophages encode diverse anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas immunity during infection of their bacterial hosts. Although detailed mechanisms have been characterized for multiple Acr proteins, an understanding of their role in phage infection biology is just emerging. Here, we review recent work in this area and propose a framework of &quot;phage autonomy&quot; to evaluate CRISPR-immune evasion strategies. During phage infection, Acr proteins are deployed by a tightly regulated &quot;fast on-fast off&quot; transcriptional burst, which is necessary, but insufficient, for CRISPR-Cas inactivation. Instead of a single phage shutting down CRISPR-Cas immunity, a community of acr-carrying phages cooperate to suppress bacterial immunity, displaying low phage autonomy. Enzymatic Acr proteins with novel mechanisms have been recently revealed and are predicted to enhance phage autonomy, while phage DNA protective measures offer the highest phage autonomy observed. These varied Acr mechanisms and strengths also have unexpected impacts on the bacterial populations and competing phages.},
}
@article {pmid33443157,
year = {2021},
author = {Kurtz, S and Lucas-Hahn, A and Schlegelberger, B and Göhring, G and Niemann, H and Mettenleiter, TC and Petersen, B},
title = {Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {2},
pages = {},
doi = {10.1073/pnas.2008743118},
pmid = {33443157},
issn = {1091-6490},
abstract = {The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located &quot;high mobility group&quot; (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.},
}
@article {pmid33441159,
year = {2021},
author = {Scott, TA and Morris, KV},
title = {Designer nucleases to treat malignant cancers driven by viral oncogenes.},
journal = {Virology journal},
volume = {18},
number = {1},
pages = {18},
pmid = {33441159},
issn = {1743-422X},
support = {R01 113407-01/MH/NIMH NIH HHS/United States ; },
abstract = {Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.},
}
@article {pmid32868402,
year = {2020},
author = {Guan, J and Bondy-Denomy, J},
title = {Intracellular Organization by Jumbo Bacteriophages.},
journal = {Journal of bacteriology},
volume = {203},
number = {2},
pages = {},
pmid = {32868402},
issn = {1098-5530},
abstract = {Since their discovery more than 100 years ago, the viruses that infect bacteria (bacteriophages) have been widely studied as model systems. Largely overlooked, however, have been &quot;jumbo phages,&quot; with genome sizes ranging from 200 to 500 kbp. Jumbo phages generally have large virions with complex structures and a broad host spectrum. While the majority of jumbo phage genes are poorly functionally characterized, recent work has discovered many unique biological features, including a conserved tubulin homolog that coordinates a proteinaceous nucleus-like compartment that houses and segregates phage DNA. The tubulin spindle displays dynamic instability and centers the phage nucleus within the bacterial host during phage infection for optimal reproduction. The shell provides robust physical protection for the enclosed phage genomes against attack from DNA-targeting bacterial immune systems, thereby endowing jumbo phages with broad resistance. In this review, we focus on the current knowledge of the cytoskeletal elements and the specialized nuclear compartment derived from jumbo phages, and we highlight their importance in facilitating spatial and temporal organization over the viral life cycle. Additionally, we discuss the evolutionary relationships between jumbo phages and eukaryotic viruses, as well as the therapeutic potential and drawbacks of jumbo phages as antimicrobial agents in phage therapy.},
}
@article {pmid32471865,
year = {2020},
author = {Das, S and Banerjee, A and Kamran, M and Ejazi, SA and Asad, M and Ali, N and Chakrabarti, S},
title = {A chemical inhibitor of heat shock protein 78 (HSP78) from Leishmania donovani represents a potential antileishmanial drug candidate.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {29},
pages = {9934-9947},
pmid = {32471865},
issn = {1083-351X},
mesh = {Animals ; Antiprotozoal Agents/*pharmacology ; CRISPR-Cas Systems ; Cricetinae ; Dinucleoside Phosphates/*pharmacology ; Gene Knockout Techniques ; Heat-Shock Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; Humans ; Leishmania donovani/genetics/*metabolism ; Leishmaniasis, Visceral/*drug therapy/genetics/metabolism ; Macrophages/metabolism/*parasitology ; Mice ; Protozoan Proteins/*antagonists &amp; inhibitors/genetics/metabolism ; },
abstract = {The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P1,P5-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.},
}
@article {pmid31959747,
year = {2020},
author = {Huang, T and Liu, Z and Zheng, Y and Feng, T and Gao, Q and Zeng, W},
title = {YTHDF2 promotes spermagonial adhesion through modulating MMPs decay via m6A/mRNA pathway.},
journal = {Cell death &amp; disease},
volume = {11},
number = {1},
pages = {37},
pmid = {31959747},
issn = {2041-4889},
mesh = {Adenosine/*analogs &amp; derivatives/metabolism ; Animals ; Apoptosis ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Adhesion/genetics ; Cell Cycle ; Cell Line ; Cell Movement ; Cell Proliferation ; Extracellular Matrix/metabolism ; Gene Deletion ; Gene Expression Regulation ; Male ; Matrix Metalloproteinases/*metabolism ; Mice, Knockout ; Phenotype ; RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Spermatogonia/*cytology/metabolism ; },
abstract = {As the foundation of male fertility, spermatogenesis is a complicated and highly controlled process. YTHDF2 plays regulatory roles in biological processes through accelerating the degradation of target mRNAs. However, the function of YTHDF2 in spermatogenesis remains elusive. Here, we knocked out Ythdf2 in mouse spermatogonia via CRISPR/Cas9, and found that depletion of Ythdf2 mainly downregulated the expression of matrix metallopeptidase (MMPs), thus affecting cell adhesion and proliferation. m6A-IP-PCR and RIP-PCR analysis showed that Mmp3, Mmp13, Adamts1 and Adamts9 were modified with m6A and simultaneously interacted with YTHDF2. Moreover, inhibition of Mmp13 partially rescued the phenotypes in Ythdf2-KO cells. Taken together, YTHDF2 regulates cell-matrix adhesion and proliferation through modulating the expression of Mmps by the m6A/mRNA degradation pathway.},
}
@article {pmid33196851,
year = {2020},
author = {Sgro, A and Blancafort, P},
title = {Epigenome engineering: new technologies for precision medicine.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {12453-12482},
pmid = {33196851},
issn = {1362-4962},
support = {R01 CA170370/CA/NCI NIH HHS/United States ; R01 DA036906/DA/NIDA NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/*genetics ; DNA Methylation/*genetics ; Epigenome/*genetics ; *Gene Editing ; Genetic Engineering ; Humans ; Precision Medicine/trends ; },
abstract = {Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more 'normal-like state', having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.},
}
@article {pmid33176167,
year = {2020},
author = {Mehta, HM and Corey, SJ},
title = {Getting Back to Normal: Correcting SCN by Universal or Precision Strikes.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2525-2526},
pmid = {33176167},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Congenital Bone Marrow Failure Syndromes ; *Hematopoietic Stem Cell Transplantation ; Mutation ; Neutropenia/congenital ; },
}
@article {pmid33152068,
year = {2020},
author = {Cooper, SE and Schwartzentruber, J and Bello, E and Coomber, EL and Bassett, AR},
title = {Screening for functional transcriptional and splicing regulatory variants with GenIE.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {e131},
pmid = {33152068},
issn = {1362-4962},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Alleles ; Alternative Splicing/genetics ; Alzheimer Disease/*genetics/pathology/therapy ; CRISPR-Cas Systems/genetics ; Clusterin/*genetics ; Enhancer Elements, Genetic/*genetics ; Gene Editing ; Genetic Variation/genetics ; Genome-Wide Association Study ; Humans ; Induced Pluripotent Stem Cells/metabolism/transplantation ; Mutation ; Polymorphism, Single Nucleotide/genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; },
abstract = {Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.},
}
@article {pmid33104788,
year = {2020},
author = {Xu, H and Wang, J and Liang, Y and Fu, Y and Li, S and Huang, J and Xu, H and Zou, W and Chen, B},
title = {TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {e127},
pmid = {33104788},
issn = {1362-4962},
mesh = {Alleles ; Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Chromatin/*genetics ; Fluorescent Antibody Technique/methods ; Gene Expression Regulation/genetics ; Gene Regulatory Networks/*genetics ; Humans ; *Molecular Imaging ; *Single-Cell Analysis ; Transcription, Genetic ; },
abstract = {A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.},
}
@article {pmid32460957,
year = {2020},
author = {Zhang, Q and Fu, Y and Thakur, C and Bi, Z and Wadgaonkar, P and Qiu, Y and Xu, L and Rice, M and Zhang, W and Almutairy, B and Chen, F},
title = {CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA.},
journal = {Biochemical and biophysical research communications},
volume = {528},
number = {1},
pages = {54-61},
pmid = {32460957},
issn = {1090-2104},
support = {P30 ES020957/ES/NIEHS NIH HHS/United States ; R01 ES028263/ES/NIEHS NIH HHS/United States ; R01 ES028335/ES/NIEHS NIH HHS/United States ; },
mesh = {Alternative Splicing/*genetics ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Dioxygenases/genetics ; Exons/genetics ; *Gene Editing ; Histone Demethylases/genetics ; Humans ; Nuclear Proteins/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA, Guide/genetics ; RNA, Messenger/genetics/metabolism ; Sequence Deletion ; },
abstract = {The technique of CRISPR-Cas9 gene editing has been widely used to specifically delete the selected target genes through generating double strand breaks (DSBs) and inducing insertion and/or deletion (indel) of the genomic DNAs in the cells. We recently applied this technique to disrupt mineral dust-induced gene (mdig), a potential oncogene as previously reported, by single guide RNA (sgRNA) targeting the third exon of mdig gene in several cell types, including human bronchial epithelial cell line BEAS-2B, lung cancer cell line A549, and human triple negative breast cancer cell line MDA-MB-231 cells. In addition to the successful knockout of mdig gene in these cells, we unexpectedly noted generation of several alternatively spliced mdig mRNAs. Amplification of the mdig mRNAs during the screening of knockout clones by reverse transcription-polymerase chain reaction (RT-PCR) and the subsequent sanger sequencing of DNA revealed deletion and alternative splicing of mdig mRNAs induced by CRISPR-Cas9 gene editing. The most common deletions include nine and twenty-four nucleotides deletion around the DSBs. In addition, interestingly, some mdig mRNAs showed skipping of the entire exon 3, or alternative splicing between exon 2 and exon 8 using the new donor and accept splicing sites, leading to deletion of exons 3, 4, 5, 6, and 7. Accordingly, cautions should be taken when using CRISPR-Cas9 strategy to edit human genes due to the unintended alterative splicing of the target mRNAs. It is very likely that new proteins, some of which may be highly oncogenic, may be generated from CRISPR-Cas9 gene editing.},
}
@article {pmid32354746,
year = {2020},
author = {Chen, X and Gao, YQ and Zheng, YY and Wang, W and Wang, P and Liang, J and Zhao, W and Tao, T and Sun, J and Wei, L and Li, Y and Zhou, Y and Gan, Z and Zhang, X and Chen, HQ and Zhu, MS},
title = {The intragenic microRNA miR199A1 in the dynamin 2 gene contributes to the pathology of X-linked centronuclear myopathy.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {26},
pages = {8656-8667},
pmid = {32354746},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Dynamin II/analysis/*genetics ; Female ; Longevity ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/analysis/*genetics ; Muscle Strength ; Muscle, Skeletal/metabolism/pathology ; Myopathies, Structural, Congenital/*genetics/pathology ; },
abstract = {Mutations in the myotubularin 1 (MTM1) gene can cause the fatal disease X-linked centronuclear myopathy (XLCNM), but the underlying mechanism is incompletely understood. In this report, using an Mtm1-/y disease model, we found that expression of the intragenic microRNA miR-199a-1 is up-regulated along with that of its host gene, dynamin 2 (Dnm2), in XLCNM skeletal muscle. To assess the role of miR-199a-1 in XLCNM, we crossed miR-199a-1-/- with Mtm1-/y mice and found that the resultant miR-199a-1-Mtm1 double-knockout mice display markers of improved health, as evidenced by lifespans prolonged by 30% and improved muscle strength and histology. Mechanistic analyses showed that miR-199a-1 directly targets nonmuscle myosin IIA (NM IIA) expression and, hence, inhibits muscle postnatal development as well as muscle maturation. Further analysis revealed that increased expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) up-regulates Dnm2/miR-199a-1 expression in XLCNM muscle. Our results suggest that miR-199a-1 has a critical role in XLCNM pathology and imply that this microRNA could be targeted in therapies to manage XLCNM.},
}
@article {pmid32245271,
year = {2020},
author = {Vidyanti, AN and Hsieh, JY and Lin, KJ and Fang, YC and Setyopranoto, I and Hu, CJ},
title = {Role of HMGB1 in an Animal Model of Vascular Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion.},
journal = {International journal of molecular sciences},
volume = {21},
number = {6},
pages = {},
pmid = {32245271},
issn = {1422-0067},
support = {MOST 106-2314-B-038-038-MY2//Ministry of Science and Technology, Taiwan/ ; },
mesh = {Amyloid beta-Peptides/metabolism ; Animals ; Behavior Rating Scale ; Brain Ischemia/diagnostic imaging/genetics/*metabolism/physiopathology ; CRISPR-Cas Systems ; Carotid Stenosis ; *Cerebrovascular Circulation ; Chronic Disease ; Dementia, Vascular/physiopathology ; Disease Models, Animal ; Gene Knockout Techniques ; HMGB1 Protein/genetics/*metabolism ; Hippocampus/diagnostic imaging/pathology/physiopathology ; Interleukin-1beta/metabolism ; Interleukin-6/metabolism ; Magnetic Resonance Imaging ; Male ; Mice ; Mice, Inbred C57BL ; Psychomotor Performance ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {The pathophysiology of vascular cognitive impairment (VCI) is associated with chronic cerebral hypoperfusion (CCH). Increased high-mobility group box protein 1 (HMGB1), a nonhistone protein involved in injury and inflammation, has been established in the acute phase of CCH. However, the role of HMGB1 in the chronic phase of CCH remains unclear. We developed a novel animal model of CCH with a modified bilateral common carotid artery occlusion (BCCAO) in C57BL/6 mice. Cerebral blood flow (CBF) reduction, the expression of HMGB1 and its proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, and IL-6), and brain pathology were assessed. Furthermore, we evaluated the effect of HMGB1 suppression through bilateral intrahippocampus injection with the CRISPR/Cas9 knockout plasmid. Three months after CCH induction, CBF decreased to 30-50% with significant cognitive decline in BCCAO mice. The 7T-aMRI showed hippocampal atrophy, but amyloid positron imaging tomography showed nonsignificant amyloid-beta accumulation. Increased levels of HMGB1, TNF-α, IL-1β, and IL-6 were observed 3 months after BCCAO. HMGB1 suppression with CRISPR/Cas9 knockout plasmid restored TNF-α, IL-1β, and IL-6 and attenuated hippocampal atrophy and cognitive decline. We believe that HMGB1 plays a pivotal role in CCH-induced VCI pathophysiology and can be a potential therapeutic target of VCI.},
}
@article {pmid33391497,
year = {2021},
author = {Wang, L and Zhou, J and Wang, Q and Wang, Y and Kang, C},
title = {Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein.},
journal = {Theranostics},
volume = {11},
number = {2},
pages = {649-664},
pmid = {33391497},
issn = {1838-7640},
mesh = {Antiviral Agents/*administration &amp; dosage ; COVID-19/*drug therapy/virology ; CRISPR-Cas Systems/genetics ; Computational Biology ; Drug Evaluation, Preclinical ; Genetic Vectors/administration &amp; dosage/genetics ; Hep G2 Cells ; Humans ; Molecular Docking Simulation ; RNA, Guide/*genetics ; SARS-CoV-2/genetics ; Sequence Homology, Amino Acid ; Spike Glycoprotein, Coronavirus/*genetics ; },
abstract = {The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.},
}
@article {pmid33303323,
year = {2021},
author = {Dronina, J and Bubniene, US and Ramanavicius, A},
title = {The application of DNA polymerases and Cas9 as representative of DNA-modifying enzymes group in DNA sensor design (review).},
journal = {Biosensors &amp; bioelectronics},
volume = {175},
number = {},
pages = {112867},
doi = {10.1016/j.bios.2020.112867},
pmid = {33303323},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; COVID-19/*diagnosis/genetics/virology ; CRISPR-Cas Systems/genetics ; Diagnostic Tests, Routine ; Humans ; RNA, Viral/genetics/*isolation &amp; purification ; SARS-CoV-2/*isolation &amp; purification/pathogenicity ; },
abstract = {Rapid detection of nucleic acids (DNA or RNA) by inexpensive, selective, accurate, and highly sensitive methods is very important for biosensors. DNA-sensors based on DNA-modifying enzymes for fast determination and monitoring of pathogenic (Zika, Dengue, SARS-Cov-2 (inducer of COVID-19), human papillomavirus, HIV, etc.) viruses and diagnosis of virus-induced diseases is a key factor of this overview. Recently, DNA-modifying enzymes (Taq DNA polymerase, Phi29 DNA polymerase) have been widely used for the diagnosis of virus or pathogenic disease by gold standard (PCR, qPCR, RT-qPCR) methods, therefore, alternative methods have been reviewed. The main mechanisms of DNA metabolism (replication cycle, amplification) and the genomeediting tool CRISPR-Cas9 are purposefully discussed in order to address strategic possibility to design DNA-sensors based on immobilized DNA-enzymes. However, the immobilization of biologically active proteins on a gold carrier technique with the ability to detect viral or bacterial nucleic acids is individual for each DNA-modifying enzyme group, due to a different number of active sites, C and N terminal locations and arrangement, therefore, individual protocols based on the 'masking' of active sites should be elaborated for each enzyme.},
}
@article {pmid32534122,
year = {2020},
author = {Li, G and Zhang, X and Wang, H and Liu, D and Li, Z and Wu, Z and Yang, H},
title = {Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors.},
journal = {The international journal of biochemistry &amp; cell biology},
volume = {125},
number = {},
pages = {105790},
doi = {10.1016/j.biocel.2020.105790},
pmid = {32534122},
issn = {1878-5875},
mesh = {Acetylation ; Animals ; Benzofurans/*pharmacology ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; Cells, Cultured ; Chromatin Immunoprecipitation ; DNA End-Joining Repair/*drug effects ; DNA Repair/drug effects ; Gene Editing/*methods ; HEK293 Cells ; Histone Deacetylase Inhibitors/*pharmacology ; Humans ; Hydroxamic Acids/*pharmacology ; Ku Autoantigen/metabolism ; Poly (ADP-Ribose) Polymerase-1/metabolism ; Rad51 Recombinase/genetics/metabolism ; Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; Recombinational DNA Repair/*drug effects ; Swine ; },
abstract = {Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.},
}
@article {pmid32470463,
year = {2020},
author = {Strezoska, Ž and Dickerson, SM and Maksimova, E and Chou, E and Gross, MM and Hemphill, K and Hardcastle, T and Perkett, M and Stombaugh, J and Miller, GW and Anderson, EM and Vermeulen, A and Smith, AVB},
title = {CRISPR-mediated transcriptional activation with synthetic guide RNA.},
journal = {Journal of biotechnology},
volume = {319},
number = {},
pages = {25-35},
doi = {10.1016/j.jbiotec.2020.05.005},
pmid = {32470463},
issn = {1873-4863},
mesh = {Animals ; Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mice ; NIH 3T3 Cells ; *RNA, Guide ; Transcriptional Activation/*genetics ; },
abstract = {The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.},
}
@article {pmid32446977,
year = {2020},
author = {Tyagi, S and Kumar, R and Das, A and Won, SY and Shukla, P},
title = {CRISPR-Cas9 system: A genome-editing tool with endless possibilities.},
journal = {Journal of biotechnology},
volume = {319},
number = {},
pages = {36-53},
doi = {10.1016/j.jbiotec.2020.05.008},
pmid = {32446977},
issn = {1873-4863},
mesh = {Animals ; Biomedical Research ; *CRISPR-Cas Systems ; *Gene Editing/ethics/methods/standards ; Humans ; Plants, Genetically Modified ; },
abstract = {The discovery of CRISPR: Cas9 and its application as a powerful gene-editing tool has transformed the world of basic and applied science, especially the molecular biology dome. Also, the smooth, quick, flexible, and very efficient nature of this technology has enabled the biologists to alter the genome of prokaryotes to complex eukaryotic systems, including plants and animals. Using CRISPR and associated tools, investigation, control, and modification of significant biological events have been more accessible than before. These biological scissors are now being used to accelerate breeding programs of crop and livestock, engineer new antimicrobials, and control disease-carrying pathogens. However, like other techniques, these cutters emerged as a double-edged sword and put several challenges to the scientific society. Here in this review article, we summarized the beneficial application of the CRISPR: Cas9 system and unsafe perception to the society if handled carelessly. We also discussed the limitations and ethical issues related to CRISPR: Cas9 technology.},
}
@article {pmid32408895,
year = {2020},
author = {Thom, CS and Jobaliya, CD and Lorenz, K and Maguire, JA and Gagne, A and Gadue, P and French, DL and Voight, BF},
title = {Tropomyosin 1 genetically constrains in vitro hematopoiesis.},
journal = {BMC biology},
volume = {18},
number = {1},
pages = {52},
pmid = {32408895},
issn = {1741-7007},
support = {R56 DK101478/DK/NIDDK NIH HHS/United States ; R01 HL130698/HL/NHLBI NIH HHS/United States ; T32HD043021//National Institute of Child Health and Human Development/International ; R01 DK101478/DK/NIDDK NIH HHS/United States ; R01DK101478/DK/NIDDK NIH HHS/United States ; T32 HD043021/HD/NICHD NIH HHS/United States ; },
mesh = {Blood Platelets/*metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Hematopoiesis/*genetics ; Hematopoietic Stem Cells/*metabolism ; Humans ; In Vitro Techniques ; Tropomyosin/deficiency/*metabolism ; },
abstract = {BACKGROUND: Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits.<br><br>RESULTS: Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields.<br><br>CONCLUSIONS: Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.},
}
@article {pmid31859532,
year = {2020},
author = {Martin, J and Free, T},
title = {A look back at 2019 in BioTechniques.},
journal = {BioTechniques},
volume = {68},
number = {1},
pages = {2-3},
doi = {10.2144/btn-2019-0164},
pmid = {31859532},
issn = {1940-9818},
mesh = {CRISPR-Cas Systems ; Humans ; *Polymerase Chain Reaction ; *Serial Publications ; Social Media ; Urine Specimen Collection/*methods ; },
}
@article {pmid33428981,
year = {2021},
author = {Safari, F and Afarid, M and Rastegari, B and Haghighi, AB and Barekati-Mowahed, M and Behbahani, AB},
title = {CRISPR systems: Novel approaches for detection and combating COVID-19.},
journal = {Virus research},
volume = {},
number = {},
pages = {198282},
doi = {10.1016/j.virusres.2020.198282},
pmid = {33428981},
issn = {1872-7492},
abstract = {Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than fifty million people worldwide with near 1.3 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.},
}
@article {pmid33428900,
year = {2021},
author = {Palaz, F and Kerem Kalkan, A and Tozluyurt, A and Ozsoz, M},
title = {CRISPR-based tools: alternative methods for the diagnosis of COVID-19.},
journal = {Clinical biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.clinbiochem.2020.12.011},
pmid = {33428900},
issn = {1873-2933},
abstract = {The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spread all over the world rapidly and caused a global pandemic. To prevent the virus from spreading to more individuals, it is of great importance to identify and isolate infected individuals through testing. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard method for the diagnosis of coronavirus disease (COVID-19) worldwide. However, performing RT-qPCR is limited to centralized laboratories because of the need for sophisticated laboratory equipment and skilled personnel. Further, it can sometimes give false negative or uncertain results. Recently, new methods have been developed for nucleic acid detection and pathogen diagnosis using CRISPR-Cas systems. These methods present rapid and cost-effective diagnostic platforms that provide high sensitivity and specificity without the need for complex instrumentation. Using the CRISPR-based SARS-CoV-2 detection methods, it is possible to increase the number of daily tests in existing laboratories, reduce false negative or uncertain result rates obtained with RT-qPCR, and perform testing in resource-limited settings or at points of need where performing RT-qPCR is not feasible. Here, we briefly describe the RT-qPCR method, and discuss its limitations in meeting the current diagnostic needs. We explain how the unique properties of various CRISPR-associated enzymes are utilized for nucleic acid detection and pathogen diagnosis. Then, we highlight the important features of CRISPR-based diagnostic methods developed for SARS-CoV-2 detection. Finally, we examine the advantages and limitations of these methods, and discuss how they can contribute to improving the efficiency of the current testing systems for combating SARS-CoV-2.},
}
@article {pmid33425987,
year = {2020},
author = {Srivastava, S and Upadhyay, DJ and Srivastava, A},
title = {Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks.},
journal = {Frontiers in molecular biosciences},
volume = {7},
number = {},
pages = {582499},
doi = {10.3389/fmolb.2020.582499},
pmid = {33425987},
issn = {2296-889X},
abstract = {Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization.},
}
@article {pmid33332350,
year = {2020},
author = {House, NCM and Parasuram, R and Layer, JV and Price, BD},
title = {Site-specific targeting of a light activated dCas9-KillerRed fusion protein generates transient, localized regions of oxidative DNA damage.},
journal = {PloS one},
volume = {15},
number = {12},
pages = {e0237759},
pmid = {33332350},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/*genetics ; DNA Breaks, Double-Stranded ; DNA Damage/*genetics ; DNA Repair/*genetics ; Endonucleases/genetics ; Genome/genetics ; HEK293 Cells ; Humans ; Light ; Oxidative Stress/*genetics ; RNA, Guide/genetics ; },
abstract = {DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in &quot;clustered&quot; oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in &quot;real time&quot; by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.},
}
@article {pmid33046744,
year = {2020},
author = {Kang, D and Shin, W and Yoo, H and Kim, S and Lee, S and Rhee, K},
title = {Cep215 is essential for morphological differentiation of astrocytes.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {17000},
pmid = {33046744},
issn = {2045-2322},
mesh = {Animals ; Astrocytes/*physiology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cell Differentiation ; Cell Line ; Mice ; Microtubules/*metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; Neuroglia/*physiology ; RNA, Small Interfering/genetics ; },
abstract = {Cep215 (also known as Cdk5rap2) is a centrosome protein which is involved in microtubule organization. Cep215 is also placed at specific subcellular locations and organizes microtubules outside the centrosome. Here, we report that Cep215 is involved in morphological differentiation of astrocytes. Cep215 was specifically localized at the glial processes as well as centrosomes in developing astrocytes. Morphological differentiation of astrocytes was suppressed in the Cep215-deleted P19 cells and in the Cep215-depleted embryonic hippocampal culture. We confirm that the microtubule organizing function of Cep215 is critical for the glial process formation. However, Cep215 is not involved in the regulation of cell proliferation nor cell specification. Based on the results, we propose that Cep215 organizes microtubules for glial process formation during astrocyte differentiation.},
}
@article {pmid32876764,
year = {2020},
author = {Zhao, N and Li, L and Luo, G and Xie, S and Lin, Y and Han, S and Huang, Y and Zheng, S},
title = {Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.},
journal = {Journal of industrial microbiology &amp; biotechnology},
volume = {47},
number = {8},
pages = {599-608},
doi = {10.1007/s10295-020-02304-5},
pmid = {32876764},
issn = {1476-5535},
support = {31671840//National Natural Science Foundation of China/ ; 2018YFA0901700//National Key R&amp;D Program of China/ ; 2019M661676//Postdoctoral Research Foundation of China/ ; },
mesh = {CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Corynebacterium glutamicum/enzymology/*genetics/metabolism ; DNA, Bacterial/*genetics ; *Gene Deletion ; Gene Editing/*methods ; Metabolic Engineering/methods ; },
abstract = {Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.},
}
@article {pmid32788672,
year = {2020},
author = {Akkaya, M and Bansal, A and Sheehan, PW and Pena, M and Cimperman, CK and Qi, CF and Yazew, T and Otto, TD and Billker, O and Miller, LH and Pierce, SK},
title = {Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13630},
pmid = {32788672},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Extracellular Matrix/*parasitology ; Female ; Malaria, Cerebral/*parasitology ; Mice ; Mice, Inbred C57BL ; Plasmodium berghei/*genetics/growth &amp; development/physiology ; *Polymorphism, Single Nucleotide ; Protozoan Proteins/antagonists &amp; inhibitors/*genetics ; Virulence Factors/antagonists &amp; inhibitors/*genetics ; },
abstract = {Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.},
}
@article {pmid32651405,
year = {2020},
author = {Gans, I and Hartig, EI and Zhu, S and Tilden, AR and Hutchins, LN and Maki, NJ and Graber, JH and Coffman, JA},
title = {Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11415},
pmid = {32651405},
issn = {2045-2322},
support = {R03 HD099468/HD/NICHD NIH HHS/United States ; P20 GM104318/GM/NIGMS NIH HHS/United States ; P20 GM103423/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; *Frameshift Mutation ; Gene Deletion ; Gene Expression Regulation ; Homozygote ; Humans ; Hydrocortisone/metabolism ; Inflammation ; Kruppel-Like Transcription Factors/*metabolism ; Larva ; Mutation ; RNA-Seq ; Receptors, Glucocorticoid/*metabolism ; Receptors, Mineralocorticoid/metabolism ; Signal Transduction ; *Transcriptome ; Up-Regulation ; Zebrafish/genetics ; Zebrafish Proteins/*metabolism ; },
abstract = {The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR369- larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.},
}
@article {pmid32403926,
year = {2020},
author = {Modell, AE and Siriwardena, SU and Choudhary, A},
title = {A Jumbo Phage Forms a Nucleus-like Compartment to Evade Bacterial Defense Systems.},
journal = {Biochemistry},
volume = {59},
number = {20},
pages = {1869-1870},
doi = {10.1021/acs.biochem.0c00273},
pmid = {32403926},
issn = {1520-4995},
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Viral ; Immune System ; },
}
@article {pmid32347717,
year = {2020},
author = {Lu, M and Tokuyasu, TA},
title = {CRISPR-Cas13-Based RNA-Interacting Protein Detection in Living Cells.},
journal = {Biochemistry},
volume = {59},
number = {19},
pages = {1791-1792},
doi = {10.1021/acs.biochem.0c00281},
pmid = {32347717},
issn = {1520-4995},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA/genetics ; },
}
@article {pmid32250930,
year = {2020},
author = {Wu, H and Qian, C and Wu, C and Wang, Z and Wang, D and Ye, Z and Ping, J and Wu, J and Ji, F},
title = {End-point dual specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor.},
journal = {Biosensors &amp; bioelectronics},
volume = {157},
number = {},
pages = {112153},
doi = {10.1016/j.bios.2020.112153},
pmid = {32250930},
issn = {1873-4235},
mesh = {Biosensing Techniques/instrumentation/*methods ; *CRISPR-Cas Systems ; Equipment Design ; Food Safety ; Lectins/genetics ; Molecular Diagnostic Techniques/instrumentation/methods ; Nucleic Acid Amplification Techniques/instrumentation/methods ; Plants, Genetically Modified/*genetics ; Polymerase Chain Reaction/instrumentation/methods ; Soybeans/*genetics ; Zea mays/*genetics ; },
abstract = {A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready@). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc.},
}
@article {pmid32208677,
year = {2020},
author = {Ramesh, A and Ong, T and Garcia, JA and Adams, J and Wheeldon, I},
title = {Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica.},
journal = {ACS synthetic biology},
volume = {9},
number = {4},
pages = {967-971},
doi = {10.1021/acssynbio.9b00498},
pmid = {32208677},
issn = {2161-5063},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal/*genetics ; Genome, Fungal/genetics ; RNA, Guide/*genetics ; Yarrowia/*genetics ; },
abstract = {Yarrowia lipolytica has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation. Truncating guide RNA spacer length to 16 nt inhibited nuclease activity but not binding to the target loci, enabling gene activation and repression with Cpf1-fused transcriptional regulators. Gene repression was demonstrated using a Cpf1-Mxi1 fusion achieving a 7-fold reduction in mRNA, while CRISPR-activation with Cpf1-VPR increased hrGFP expression by 10-fold. High efficiency disruptions were achieved with gRNAs 23-25 bp in length, and efficiency and repression levels were maintained with multiplexed expression of truncated and full-length gRNAs. The developed CRISPR-Cpf1 system should prove useful in metabolic engineering, genome wide screening, and functional genomics studies.},
}
@article {pmid32160465,
year = {2020},
author = {Liow, LT and Go, MK and Chang, MW and Yew, WS},
title = {Toolkit Development for Cyanogenic and Gold Biorecovery Chassis Chromobacterium violaceum.},
journal = {ACS synthetic biology},
volume = {9},
number = {4},
pages = {953-961},
doi = {10.1021/acssynbio.0c00064},
pmid = {32160465},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/genetics ; *Chromobacterium/genetics/metabolism ; Cyanides/isolation &amp; purification/metabolism ; Drug Resistance, Bacterial/genetics ; Electronic Waste ; Genetic Engineering/*methods ; Gold/isolation &amp; purification/metabolism ; Promoter Regions, Genetic/genetics ; Synthetic Biology/*methods ; },
abstract = {Chromobacterium violaceum has been of interest recently due to its cyanogenic ability and its potential role in environmental sustainability via the biorecovery of gold from electronic waste. However, as with many nonmodel bacteria, there are limited genetic tools to implement the use of this Gram-negative chassis in synthetic biology. We propose a system that involves assaying spontaneous antibiotic resistances and using broad host range vectors to develop episomal vectors for nonmodel Gram-negative bacteria. These developed vectors can subsequently be used to characterize inducible promoters for gene expressions and implementing CRISPRi to inhibit endogenous gene expression for further studies. Here, we developed the first episomal genetic toolkit for C. violaceum consisting of two origins of replication, three antibiotic resistance genes, and four inducible promoter systems. We examined the occurrences of spontaneous resistances of the bacterium to the chosen selection markers to prevent incidences of false positives. We also tested broad host range vectors from four different incompatibility groups and characterized four inducible promoter systems, which potentially can be applied in other Gram-negative nonmodel bacteria. CRISPRi was also implemented to inhibit violacein pigment production in C. violaceum. This systematic toolkit will aid future genetic circuitry building in this chassis and other nonmodel bacteria for synthetic biology and biotechnological applications.},
}
@article {pmid32142605,
year = {2020},
author = {Wrist, A and Sun, W and Summers, RM},
title = {The Theophylline Aptamer: 25 Years as an Important Tool in Cellular Engineering Research.},
journal = {ACS synthetic biology},
volume = {9},
number = {4},
pages = {682-697},
doi = {10.1021/acssynbio.9b00475},
pmid = {32142605},
issn = {2161-5063},
mesh = {*Aptamers, Nucleotide ; CRISPR-Cas Systems ; *Cell Engineering ; Cell Line ; Humans ; RNA Interference ; *Riboswitch ; Synthetic Biology ; *Theophylline ; },
abstract = {The theophylline aptamer was isolated from an oligonucleotide library in 1994. Since that time, the aptamer has found wide utility, particularly in synthetic biology, cellular engineering, and diagnostic applications. The primary application of the theophylline aptamer is in the construction and characterization of synthetic riboswitches for regulation of gene expression. These riboswitches have been used to control cellular motility, regulate carbon metabolism, construct logic gates, screen for mutant enzymes, and control apoptosis. Other applications of the theophylline aptamer in cellular engineering include regulation of RNA interference and genome editing through CRISPR systems. Here we describe the uses of the theophylline aptamer for cellular engineering over the past 25 years. In so doing, we also highlight important synthetic biology applications to control gene expression in a ligand-dependent manner.},
}
@article {pmid31953347,
year = {2020},
author = {Zhang, L and He, A and Chen, B and Bi, J and Chen, J and Guo, D and Qian, Y and Wang, W and Shi, T and Zhao, Z and Shi, J and An, W and Attenello, F and Lu, W},
title = {A HOTAIR regulatory element modulates glioma cell sensitivity to temozolomide through long-range regulation of multiple target genes.},
journal = {Genome research},
volume = {30},
number = {2},
pages = {155-163},
pmid = {31953347},
issn = {1549-5469},
mesh = {Antineoplastic Agents, Alkylating/pharmacology ; Base Sequence ; CRISPR-Cas Systems/genetics ; Calcium-Binding Proteins/*genetics ; Carrier Proteins/*genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Regulatory Networks/genetics ; Glioma/*drug therapy/genetics/pathology ; Humans ; Neoplasm Proteins/genetics ; RNA, Long Noncoding/*genetics ; Temozolomide/*pharmacology ; Transcription Factors/*genetics ; },
abstract = {Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.},
}
@article {pmid31944664,
year = {2020},
author = {Ou, B and Jiang, B and Jin, D and Yang, Y and Zhang, M and Zhang, D and Zhao, H and Xu, M and Song, H and Wu, W and Chen, M and Lu, T and Huang, J and Seo, H and Garcia, C and Zheng, W and Guo, W and Lu, Y and Jiang, Y and Yang, S and Kaushik, RS and Li, X and Zhang, W and Zhu, G},
title = {Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.},
journal = {ACS synthetic biology},
volume = {9},
number = {2},
pages = {412-426},
doi = {10.1021/acssynbio.9b00430},
pmid = {31944664},
issn = {2161-5063},
mesh = {Adhesins, Escherichia coli/immunology ; Animals ; Antibodies, Bacterial/blood/immunology ; Bacterial Adhesion ; CRISPR-Cas Systems/*genetics ; Cell Line ; *Chromosomes, Bacterial ; Enterotoxigenic Escherichia coli/*genetics/immunology ; Epithelial Cells/cytology/metabolism ; Escherichia coli Proteins/genetics/immunology ; Female ; Immunoglobulin G/immunology/metabolism ; Mice ; Mice, Inbred BALB C ; Multigene Family ; Swine ; },
abstract = {F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.},
}
@article {pmid31943709,
year = {2020},
author = {},
title = {Mucopolysaccharidosis Type I Phenotypically Corrected with Edited Hematopoietic Stem Cells: Instead of altering the IDUA gene, a protein was inserted in a repurposable place in the genome known as a &quot;safe harbor locus&quot;.},
journal = {American journal of medical genetics. Part A},
volume = {182},
number = {2},
pages = {275-276},
doi = {10.1002/ajmg.a.61223},
pmid = {31943709},
issn = {1552-4833},
mesh = {Animals ; Antigens, CD34/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/metabolism ; Humans ; Iduronidase/*genetics ; Mice ; Mucopolysaccharidosis I/*genetics/pathology/therapy ; Phenotype ; Receptors, CCR5/*genetics ; },
}
@article {pmid31891494,
year = {2020},
author = {Fan, J and Liu, Y and Liu, L and Huang, Y and Li, X and Huang, W},
title = {A Multifunction Lipid-Based CRISPR-Cas13a Genetic Circuit Delivery System for Bladder Cancer Gene Therapy.},
journal = {ACS synthetic biology},
volume = {9},
number = {2},
pages = {343-355},
doi = {10.1021/acssynbio.9b00349},
pmid = {31891494},
issn = {2161-5063},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Disease Models, Animal ; Gene Editing ; *Genetic Therapy ; Humans ; Infrared Rays ; Liposomes/chemistry/*metabolism ; Mice ; Mice, Nude ; Photothermal Therapy ; Plasmids/genetics/metabolism ; Transfection ; Urinary Bladder Neoplasms/pathology/therapy ; Vascular Endothelial Growth Factor Receptor-2/genetics/metabolism ; },
abstract = {The treatment of bladder cancer has recently shown minimal progress. Gene therapy mediated by CRISPR provides a new option for bladder cancer treatment. In this study, we developed a versatile liposome system to deliver the CRISPR-Cas13a gene circuits into bladder cancer cells. After in vitro studies and intravesical perfusion studies in mice, this system showed five advantages: (1) CRISPR-Cas13a, a transcriptional targeting and cleavage tool for gene expression editing, did not affect the stability of the cell genome; (2) the prepared liposome systems were targeted to hVEGFR2, which is always highly expressed in bladder cancer cells; (3) the CRISPR-Cas13a sequence was driven by an artificial tumor specific promoter to achieve further targeting; (4) a near-infrared photosensitizer released using near-infrared light was introduced to control the delivery system; and (5) the plasmids were constructed with three crRNA tandem sequences to achieve multiple targeting and wider therapeutic results. This tumor cell targeting lipid delivery system with near-infrared laser-controlled ability provided a versatile strategy for CRISPR-Cas13a based gene therapy of bladder cancer.},
}
@article {pmid31874862,
year = {2020},
author = {Katayama, S and Sato, K and Nakazawa, T},
title = {In vivo and in vitro knockout system labelled using fluorescent protein via microhomology-mediated end joining.},
journal = {Life science alliance},
volume = {3},
number = {1},
pages = {},
pmid = {31874862},
issn = {2575-1077},
mesh = {Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Carnitine O-Acetyltransferase/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; DNA End-Joining Repair/*genetics ; Dependovirus/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Gene Knockout Techniques/*methods ; Genetic Vectors ; Green Fluorescent Proteins/*genetics ; Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NIH 3T3 Cells ; Retinal Ganglion Cells/metabolism ; Staining and Labeling/*methods ; Staphylococcus aureus/enzymology ; Transfection ; },
abstract = {Gene knockout is important for understanding gene function and genetic disorders. The CRISPR/Cas9 system has great potential to achieve this purpose. However, we cannot distinguish visually whether a gene is knocked out and in how many cells it is knocked out among a population of cells. Here, we developed a new system that enables the labelling of knockout cells with fluorescent protein through microhomology-mediated end joining-based knock-in. Using a combination with recombinant adeno-associated virus, we delivered our system into the retina, where the expression of Staphylococcus aureus Cas9 was driven by a retina ganglion cell (RGC)-specific promoter, and knocked out carnitine acetyltransferase (CAT). We evaluated RGCs and revealed that CAT is required for RGC survival. Furthermore, we applied our system to Keap1 and confirmed that Keap1 is not expressed in fluorescently labelled cells. Our system provides a promising framework for cell type-specific genome editing and fluorescent labelling of gene knockout based on knock-in.},
}
@article {pmid31758450,
year = {2020},
author = {Jacot, D and Soldati-Favre, D},
title = {CRISPR/Cas9-Mediated Generation of Tetracycline Repressor-Based Inducible Knockdown in Toxoplasma gondii.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2071},
number = {},
pages = {125-141},
doi = {10.1007/978-1-4939-9857-9_7},
pmid = {31758450},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryptosporidium/genetics/pathogenicity ; Gene Knockout Techniques ; Genome, Protozoan/genetics ; Mutagenesis, Site-Directed ; Protein Stability ; Temperature ; Toxoplasma/genetics/*pathogenicity ; },
abstract = {The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii.},
}
@article {pmid33422414,
year = {2021},
author = {Behbahani, RG and Danyaei, A and Teimoori, A and Neisi, N and Tahmasbi, MJ},
title = {Breast cancer radioresistance may be overcome by osteopontin gene knocking out with CRISPR/Cas9 technique.},
journal = {Cancer radiotherapie : journal de la Societe francaise de radiotherapie oncologique},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.canrad.2020.08.048},
pmid = {33422414},
issn = {1769-6658},
abstract = {PURPOSE: Osteopontin (OPN) is a phosphoglycoprotein, with a wide range of physiological and pathological roles. High expression of OPN promotes aggressive behavior, causes poor prognosis in tumor cells, and reduces the survival of patients. Since overexpression of OPN gives rise to radioresistance, the effects of the gene knock out using the CRISPR/Cas9 system in combination with radiation are emphasized.<br><br>MATERIAL AND METHODS: We used the CRISPR/Cas9 technique to knock out the OPN gene in the MDA-MB-231 cell line. After transfection, the cells were irradiated. The changes of the OPN mRNA levels, the apoptosis, and the differences in cell viability were assessed.<br><br>RESULTS: A significant reduction in the OPN expression was observed alone or along with irradiation. The knocked out gene alone increased apoptosis rate. The cell viability decreased to after knocking out of the OPN gene. The gene knocking-out combined with irradiation led to more decline of cell viability.<br><br>CONCLUSION: Our results demonstrated that after knocking out the OPN gene, the MDA-MB-231 cells showed a significant radiosensitivity. Therefore, the OPN knock out in combination with conventional radiotherapy, may become an efficient therapeutic target in the future.},
}
@article {pmid33421768,
year = {2021},
author = {Oh, SH and Lee, HJ and Ahn, MK and Jeon, MY and Yoon, JS and Jung, YJ and Kim, GN and Baek, IJ and Kim, I and Kim, KM and Sung, YH},
title = {Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA.},
journal = {Biochemical and biophysical research communications},
volume = {539},
number = {},
pages = {48-55},
doi = {10.1016/j.bbrc.2020.12.072},
pmid = {33421768},
issn = {1090-2104},
abstract = {CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.},
}
@article {pmid33421620,
year = {2021},
author = {Ansari, I and Chaturvedi, A and Chitkara, D and Singh, S},
title = {CRISPR/Cas mediated epigenome editing for cancer therapy.},
journal = {Seminars in cancer biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.semcancer.2020.12.018},
pmid = {33421620},
issn = {1096-3650},
abstract = {The understanding of the relationship between epigenetic alterations, their effects on gene expression and the knowledge that these epigenetic alterations are reversible, have opened up new therapeutic pathways for treating various diseases, including cancer. This has led the research for a better understanding of the mechanism and pathways of carcinogenesis and provided the opportunity to develop the therapeutic approaches by targeting such pathways. Epi-drugs, DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors are the best examples of epigenetic therapies with clinical applicability. Moreover, precise genome editing technologies such as CRISPR/Cas has proven their efficacy in epigenome editing, including the alteration of epigenetic markers, such as DNA methylation or histone modification. The main disadvantage with DNA gene editing technologies is off-target DNA sequence alteration, which is not an issue with epigenetic editing. It is known that cancer is linked with epigenetic alteration, and thus CRISPR/Cas system shows potential for cancer therapy via epigenome editing. This review outlines the epigenetic therapeutic approach for cancer therapy using CRISPR/Cas, from the basic understanding of cancer epigenetics to potential applications of CRISPR/Cas in treating cancer.},
}
@article {pmid33421369,
year = {2021},
author = {Workman, RE and Pammi, T and Nguyen, BTK and Graeff, LW and Smith, E and Sebald, SM and Stoltzfus, MJ and Euler, CW and Modell, JW},
title = {A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2020.12.017},
pmid = {33421369},
issn = {1097-4172},
abstract = {CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.},
}
@article {pmid33420656,
year = {2021},
author = {Yuza, K and Nagahashi, M and Ichikawa, H and Hanyu, T and Nakajima, M and Shimada, Y and Ishikawa, T and Sakata, J and Takeuchi, S and Okuda, S and Matsuda, Y and Abe, M and Sakimura, K and Takabe, K and Wakai, T},
title = {Activin a Receptor Type 2A Mutation Affects the Tumor Biology of Microsatellite Instability-High Gastric Cancer.},
journal = {Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract},
volume = {},
number = {},
pages = {},
pmid = {33420656},
issn = {1873-4626},
support = {17K10579//Japan Society for the Promotion of Science/ ; 19H03714//Japan Society for the Promotion of Science/ ; 18K19576//Japan Society for the Promotion of Science/ ; 19K09212//Japan Society for the Promotion of Science/ ; 19K09165//Japan Society for the Promotion of Science/ ; 17K10692//Japan Society for the Promotion of Science/ ; 18K08612//Japan Society for the Promotion of Science/ ; 17H04093//Japan Society for the Promotion of Science/ ; 18H04123//Japan Society for the Promotion of Science/ ; 19K22651//Japan Society for the Promotion of Science/ ; R01CA160688/CA/NCI NIH HHS/United States ; },
abstract = {BACKGROUND: Activin A receptor type 2A (ACVR2A) is one of the most frequently mutated genes in microsatellite instability-high (MSI-H) gastric cancer. However, the clinical relevance of the ACVR2A mutation in MSI-H gastric cancer patients remains unclear. The aims of this study were to explore the effect of ACVR2A mutation on the tumor behavior and to identify the clinicopathological characteristics of gastric cancer patients with ACVR2A mutations.<br><br>METHODS: An in vitro study was performed to investigate the biological role of ACVR2A via CRISPR/Cas9-mediated ACVR2A knockout MKN74 human gastric cancer cells. One hundred twenty-four patients with gastric cancer were retrospectively analyzed, and relations between MSI status, ACVR2A mutations, and clinicopathological factors were evaluated.<br><br>RESULTS: ACVR2A knockout cells showed less aggressive tumor biology than mock-transfected cells, displaying reduced proliferation, migration, and invasion (P < 0.05). MSI mutations were found in 10% (13/124) of gastric cancer patients, and ACVR2A mutations were found in 8.1% (10/124) of patients. All ACVR2A mutations were accompanied by MSI. The 5-year overall survival rates of ACVR2A wild-type patients and ACVR2A-mutated patients were 57% and 90%, respectively (P = 0.048). Multivariate analysis revealed that older age (P = 0.015), distant metastasis (P < 0.001), and ACVR2A wild-type status (P = 0.040) were independent prognostic factors for overall survival.<br><br>CONCLUSIONS: Our study demonstrated that gastric cancer patients with ACVR2A mutation have a significantly better prognosis than those without. Dysfunction of ACVR2A in MKN74 human gastric cancer cells caused less aggressive tumor biology, indicating the importance of ACVR2A in the progression of MSI-H tumors.},
}
@article {pmid33418105,
year = {2020},
author = {Das Mukhopadhyay, C and Sharma, P and Sinha, K and Rajarshi, K},
title = {Recent trends in analytical and digital techniques for the detection of the SARS-Cov-2.},
journal = {Biophysical chemistry},
volume = {270},
number = {},
pages = {106538},
doi = {10.1016/j.bpc.2020.106538},
pmid = {33418105},
issn = {1873-4200},
abstract = {The current global outbreak of COVID-19 due to SARS-CoV-2 is an unprecedented humanitarian crisis. Considering the gravity of its impact there is an immediate need to develop a detection technique that is sensitive, specific, fast, and affordable for the clinical diagnosis of the disease. Real time Polymerase Chain Reaction (RT-PCR)-based detection platforms are contemplated to be the gold standard to detect viral RNA. However, that may be susceptible to errors, and there is a risk of obtaining false results, which ultimately compromises the strategy of efficient disease management. Several modern techniques exhibiting assured results with enhanced sensitivity and specificity against the SARS-CoV-2 associated viral components or immune response against it have been developed and may be implemented. The review deals with the conventional RT-PCR detection techniques and compares them to other detection platforms viz., biosensor based detection of antigens, fluorescent or colorimetric detection systems including CRISPR-Cas 13 based SHERLOCK kit, CRISPR Cas-9 based FELUDA test kit, CRISPR DETECTR kit, Next Generation Sequencing or microarray-based kits. These modern techniques are great as a point of care detection methods but should be followed by RT PCR based detection for the confirmation of COVID-19 status.},
}
@article {pmid33412762,
year = {2020},
author = {Park, YK and Yoon, BH and Park, SJ and Kim, BK and Kim, SY},
title = {BaSDAS: a web-based pooled CRISPR-Cas9 knockout screening data analysis system.},
journal = {Genomics &amp; informatics},
volume = {18},
number = {4},
pages = {e46},
doi = {10.5808/GI.2020.18.4.e46},
pmid = {33412762},
issn = {1598-866X},
support = {2014M3C9A3068554//National Research Foundation of Korea/ ; 2017MBA9B5060884//National Research Foundation of Korea/ ; //KRIBB Research Initiative Program/ ; },
abstract = {We developed the BaSDAS (Barcode-Seq Data Analysis System), a GUI-based pooled knockout screening data analysis system, to facilitate the analysis of pooled knockout screen data easily and effectively by researchers with limited bioinformatics skills. The BaSDAS supports the analysis of various pooled screening libraries, including yeast, human, and mouse libraries, and provides many useful statistical and visualization functions with a user-friendly web interface for convenience. We expect that BaSDAS will be a useful tool for the analysis of genome-wide screening data and will support the development of novel drugs based on functional genomics information.},
}
@article {pmid32860838,
year = {2020},
author = {Das, S and Chandrasekaran, AP and Jo, KS and Ko, NR and Oh, SJ and Kim, KS and Ramakrishna, S},
title = {HAUSP stabilizes Cdc25A and protects cervical cancer cells from DNA damage response.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1867},
number = {12},
pages = {118835},
doi = {10.1016/j.bbamcr.2020.118835},
pmid = {32860838},
issn = {1879-2596},
mesh = {Animals ; CRISPR-Cas Systems ; DNA Damage/genetics ; Drug Resistance, Neoplasm/*genetics ; Female ; Flow Cytometry ; Gene Expression Regulation, Neoplastic/genetics ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Heterografts ; Humans ; Mice ; Ubiquitin-Specific Peptidase 7/*genetics ; Uterine Cervical Neoplasms/drug therapy/*genetics/pathology ; cdc25 Phosphatases/*genetics ; },
abstract = {Resistance to DNA-damaging agents is one of the main reasons for the low survival of cervical cancer patients. Previous reports have suggested that the Cdc25A oncoprotein significantly affects the level of susceptibility to DNA-damaging agents, but the molecular mechanism remains unclear. In this study, we used Western blot and flow cytometry analyses to demonstrate that the deubiquitinating enzyme HAUSP stabilizes Cdc25A protein level. Furthermore, in a co-immunoprecipitation assay, we found that HAUSP interacts with and deubiquitinates Cdc25A both exogenously and endogenously. HAUSP extends the half-life of the Cdc25A protein by circumventing turnover. HAUSP knockout in HeLa cells using the CRISPR/Cas9 system caused a significant delay in Cdc25A-mediated cell cycle progression, cell migration, and colony formation and attenuated tumor progression in a mouse xenograft model. Furthermore, HAUSP-mediated stabilization of the Cdc25A protein produced enhanced resistance to DNA-damaging agents. Overall, our study suggests that targeting Cdc25A and HAUSP could be a promising combinatorial approach to halt progression and minimize antineoplastic resistance in cervical cancer.},
}
@article {pmid32496007,
year = {2020},
author = {Zhang, W and Kataoka, M and Yen Doan, H and Wu, FT and Haga, K and Takeda, N and Muramatsu, M and Li, TC},
title = {Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection.},
journal = {Transboundary and emerging diseases},
volume = {67},
number = {6},
pages = {2849-2859},
doi = {10.1111/tbed.13655},
pmid = {32496007},
issn = {1865-1682},
support = {(JP18fk0210043, JP19fk0108102)//Japan Agency for Medical Reserach and development (AMED)/ ; 17K08090//Grabt-in-Aid for Scientific Reserarch/ ; },
mesh = {Animals ; Antibodies, Viral/blood ; Blotting, Western/veterinary ; CRISPR-Cas Systems ; Cell Line ; Electrophoresis, Polyacrylamide Gel/veterinary ; Enzyme-Linked Immunosorbent Assay/veterinary ; Feces/virology ; High-Throughput Nucleotide Sequencing/veterinary ; Immunoglobulin G/blood ; Microscopy, Electron/veterinary ; Orthoreovirus, Mammalian/genetics/immunology/*isolation &amp; purification ; Phylogeny ; Picornaviridae/*pathogenicity ; Picornaviridae Infections/*veterinary/virology ; RNA, Viral/genetics ; Reoviridae Infections/*veterinary/virology ; Swine ; Swine Diseases/*virology ; },
abstract = {Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases.},
}
@article {pmid32147886,
year = {2020},
author = {He, XY and Ren, XH and Peng, Y and Zhang, JP and Ai, SL and Liu, BY and Xu, C and Cheng, SX},
title = {Aptamer/Peptide-Functionalized Genome-Editing System for Effective Immune Restoration through Reversal of PD-L1-Mediated Cancer Immunosuppression.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {32},
number = {17},
pages = {e2000208},
doi = {10.1002/adma.202000208},
pmid = {32147886},
issn = {1521-4095},
support = {51533006//National Natural Science Foundation of China/ ; 21875169//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Apoptosis ; Aptamers, Nucleotide/*chemistry ; B7-H1 Antigen/*metabolism ; CD8-Positive T-Lymphocytes/cytology/immunology/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Gene Editing/*methods ; Humans ; Hyaluronic Acid/chemistry ; Immunosuppression ; Nanoparticles/chemistry ; Oligodeoxyribonucleotides/chemistry ; Peptides/*chemistry ; Plasmids/chemistry/metabolism ; Programmed Cell Death 1 Receptor/metabolism ; beta Catenin/deficiency/genetics/metabolism ; },
abstract = {Effective reversal of tumor immunosuppression is of critical importance in cancer therapy. A multifunctional delivery vector that can effectively deliver CRISPR-Cas9 plasmid for β-catenin knockout to reverse tumor immunosuppression is constructed. The multi-functionalized delivery vector is decorated with aptamer-conjugated hyaluronic acid and peptide-conjugated hyaluronic acid to combine the tumor cell/nuclear targeting function of AS1411 with the cell penetrating/nuclear translocation function of TAT-NLS. Due to the significantly enhanced plasmid enrichment in malignant cell nuclei, the genome editing system can induce effective β-catenin knockout and suppress Wnt/β-catenin pathway, resulting in notably downregulated proteins involved in tumor progression and immunosuppression. Programmed death-ligand 1 (PD-L1) downregulation in edited tumor cells not only releases the PD-1/PD-L1 brake to improve the cancer killing capability of CD8+ T cells, but also enhances antitumor immune responses of immune cells. This provides a facile strategy to reverse tumor immunosuppression and to restore immunosurveillance and activate anti-tumor immunity.},
}
@article {pmid31981430,
year = {2020},
author = {Chen, L and Cai, Y and Qu, M and Wang, L and Sun, H and Jiang, B and Wu, T and Liu, L and Sun, S and Wu, C and Yao, W and Yuan, S and Han, T and Hou, W},
title = {Soybean adaption to high-latitude regions is associated with natural variations of GmFT2b, an ortholog of FLOWERING LOCUS T.},
journal = {Plant, cell &amp; environment},
volume = {43},
number = {4},
pages = {934-944},
pmid = {31981430},
issn = {1365-3040},
mesh = {Adaptation, Physiological/genetics ; Arabidopsis Proteins/genetics/physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cloning, Molecular ; Flowers/growth &amp; development ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; Genetic Variation/genetics/physiology ; Geography ; Photoperiod ; Plant Proteins/*genetics/physiology ; Soybeans/genetics/growth &amp; development/*physiology ; Transcription Factors/*genetics/physiology ; Transcriptome ; },
abstract = {Day length has an important influence on flowering and growth habit in many plant species. In crops such as soybean, photoperiod sensitivity determines the geographical range over which a given cultivar can grow and flower. The soybean genome contains ~10 genes homologous to FT, a central regulator of flowering from Arabidopsis thaliana. However, the precise roles of these soybean FTs are not clearly. Here we show that one such gene, GmFT2b, promotes flowering under long-days (LDs). Overexpression of GmFT2b upregulates expression of flowering-related genes which are important in regulating flowering time. We propose a 'weight' model for soybean flowering under short-day (SD) and LD conditions. Furthermore, we examine GmFT2b sequences in 195 soybean cultivars, as well as flowering phenotypes, geographical distributions and maturity groups. We found that Hap3, a major GmFT2b haplotype, is associated with significantly earlier flowering at higher latitudes. We anticipate our assay to provide important resources for the genetic improvement of soybean, including new germplasm for soybean breeding, and also increase our understanding of functional diversity in the soybean FT gene family.},
}
@article {pmid31882406,
year = {2020},
author = {Kandul, NP and Liu, J and Buchman, A and Gantz, VM and Bier, E and Akbari, OS},
title = {Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes.},
journal = {G3 (Bethesda, Md.)},
volume = {10},
number = {2},
pages = {827-837},
pmid = {31882406},
issn = {2160-1836},
support = {DP5 OD023098/OD/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Gene Drive Technology ; Gene Editing ; *Gene Knockout Techniques ; Gene Order ; *Gene Targeting/methods ; Genetic Vectors/genetics ; Genotyping Techniques ; Models, Genetic ; Mutation ; RNA, Guide ; Zygote/metabolism ; },
abstract = {Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.},
}
@article {pmid31589320,
year = {2020},
author = {Dekkers, JF and Whittle, JR and Vaillant, F and Chen, HR and Dawson, C and Liu, K and Geurts, MH and Herold, MJ and Clevers, H and Lindeman, GJ and Visvader, JE},
title = {Modeling Breast Cancer Using CRISPR-Cas9-Mediated Engineering of Human Breast Organoids.},
journal = {Journal of the National Cancer Institute},
volume = {112},
number = {5},
pages = {540-544},
pmid = {31589320},
issn = {1460-2105},
mesh = {Animals ; Breast/cytology/*physiology ; Breast Neoplasms/*genetics/pathology ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Editing/methods ; Gene Knockout Techniques ; Genes, p53 ; Heterografts ; Humans ; Mice, Inbred NOD ; Mice, SCID ; Organoids ; PTEN Phosphohydrolase/genetics ; Retinoblastoma Binding Proteins/genetics ; Tissue Engineering/methods ; Tumor Suppressor Protein p53/genetics ; Ubiquitin-Protein Ligases/genetics ; },
abstract = {Breast cancer is characterized by histological and functional heterogeneity, posing a clinical challenge for patient treatment. Emerging evidence suggests that the distinct subtypes reflect the repertoire of genetic alterations and the target cell. However, the precise initiating events that predispose normal epithelium to neoplasia are poorly understood. Here, we demonstrate that breast epithelial organoids can be generated from human reduction mammoplasties (12 out of 12 donors), thus creating a tool to study the clonal evolution of breast cancer. To recapitulate de novo oncogenesis, we exploited clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted knockout of four breast cancer-associated tumor suppressor genes (P53, PTEN, RB1, NF1) in mammary progenitor cells from six donors. Mutant organoids gained long-term culturing capacity and formed estrogen-receptor positive luminal tumors on transplantation into mice for one out of six P53/PTEN/RB1-mutated and three out of six P53/PTEN/RB1/NF1-mutated lines. These organoids responded to endocrine therapy or chemotherapy, supporting the potential utility of this model to enhance our understanding of the molecular events that culminate in specific subtypes of breast cancer.},
}
@article {pmid33412428,
year = {2021},
author = {Liu, PF and Zhao, KR and Liu, ZJ and Wang, L and Ye, SY and Liang, GX},
title = {Cas12a-based electrochemiluminescence biosensor for target amplification-free DNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {176},
number = {},
pages = {112954},
doi = {10.1016/j.bios.2020.112954},
pmid = {33412428},
issn = {1873-4235},
abstract = {CRISPR/Cas system have drawn increasing attention in accurate and sensitive nucleic acids detection. Herein, we reported a novel Cas12a-based electrochemiluminescence biosensor for target amplification-free human papilloma virus subtype (HPV-16) DNA detection. During this detection process, Cas12a employed its two-part recognition mechanism to improve the specificity and trans-cleavage capability to achieve signal amplification, while L-Methionine stabilized gold nanoclusters (Met-AuNCs) were served as high-efficiency ECL emitters to achieve ECL signal transition. Given the unique combination of Cas12a with ECL technique, the detection limit was determined as 0.48 pM and the whole detection could be completed within 70 min. We also validated the practical application of the proposed biosensor by using undiluted human blood samples, which gives impetus to the design of new generations of CRISPR/Cas detection system beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.},
}
@article {pmid33411765,
year = {2021},
author = {Jubair, L and Lam, AK and Fallaha, S and McMillan, NAJ},
title = {CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice.},
journal = {PloS one},
volume = {16},
number = {1},
pages = {e0223288},
doi = {10.1371/journal.pone.0223288},
pmid = {33411765},
issn = {1932-6203},
abstract = {Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.},
}
@article {pmid33411517,
year = {2021},
author = {Zhou, T and Huang, M and Lin, J and Huang, R and Xing, D},
title = {High-Fidelity CRISPR/Cas13a trans-Cleavage-Triggered Rolling Circle Amplified DNAzyme for Visual Profiling of MicroRNA.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.0c03708},
pmid = {33411517},
issn = {1520-6882},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) system innovates a next-generation biosensor due to its high-fidelity, programmability, and efficient signal amplification ability. Developing a CRISPR/Cas-based visual detection system could contribute to point-of-care biomarker diagnosis. Existing CRISPR/Cas9-mediated visual detection methods are limited by the inherent properties of Cas9. Herein, we explored the trans-cleavage ability of Cas13a on ribonucleotide-bearing DNA oligo, eliminated the unavailability of the trans-cleavage substrate for subsequent polymerization reaction, and developed a homogeneous CRISPR/Cas13a-based visual detection system (termed vCas) for specific and sensitive detection of miRNA. The results indicated that vCas can provide a detection limit of 1 fM for miR-10b with single-base specificity and can be used to analyze miRNA in serum and cell extracts. Conclusively, vCas holds a great application prospective for clinical molecular diagnosis.},
}
@article {pmid33410425,
year = {2020},
author = {Sreepadmanabh, M and Sahu, AK and Chande, A},
title = {COVID-19: Advances in diagnostic tools, treatment strategies, and vaccine development.},
journal = {Journal of biosciences},
volume = {45},
number = {},
pages = {},
pmid = {33410425},
issn = {0973-7138},
abstract = {An unprecedented worldwide spread of the SARS-CoV-2 has imposed severe challenges on healthcare facilities and medical infrastructure. The global research community faces urgent calls for the development of rapid diagnostic tools, effective treatment protocols, and most importantly, vaccines against the pathogen. Pooling together expertise across broad domains to innovate effective solutions is the need of the hour. With these requirements in mind, in this review, we provide detailed critical accounts on the leading efforts at developing diagnostics tools, therapeutic agents, and vaccine candidates. Importantly, we furnish the reader with a multidisciplinary perspective on how conventional methods like serology and RT-PCR, as well as cutting-edge technologies like CRISPR/Cas and artificial intelligence/machine learning, are being employed to inform and guide such investigations. We expect this narrative to serve a broad audience of both active and aspiring researchers in the field of biomedical sciences and engineering and help inspire radical new approaches towards effective detection, treatment, and prevention of this global pandemic.},
}
@article {pmid33402545,
year = {2020},
author = {Konishi, CT and Long, C},
title = {Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases.},
journal = {Journal of biomedical research},
volume = {},
number = {},
pages = {1-15},
doi = {10.7555/JBR.34.20200105},
pmid = {33402545},
issn = {1674-8301},
abstract = {There are an estimated 10 000 monogenic diseases affecting tens of millions of individuals worldwide. The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients. CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA. The complexity of genomic insults resulting in heritable disease requires patient-specific genome editing strategies with consideration of DNA repair pathways, and CRISPR/Cas systems of different types, species, and those with additional enzymatic capacity and/or delivery methods. In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair, non-homologous end joining, microhomology-mediated end joining, and base editing to permanently correct diverse monogenic diseases.},
}
@article {pmid33402270,
year = {2020},
author = {Zhang, Y and Xi, H and Juhas, M},
title = {Biosensing Detection of the SARS-CoV-2 D614G Mutation.},
journal = {Trends in genetics : TIG},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tig.2020.12.004},
pmid = {33402270},
issn = {0168-9525},
abstract = {The emergence of a mutant strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an amino acid change from aspartate to a glycine residue at position 614 (D614G) has been reported and this mutant appears to be now dominant in the pandemic. Efficient detection of the SARS-CoV-2 D614G mutant by biosensing technologies is therefore crucial for the control of the pandemic.},
}
@article {pmid33401751,
year = {2021},
author = {Taliansky, M and Samarskaya, V and Zavriev, SK and Fesenko, I and Kalinina, NO and Love, AJ},
title = {RNA-Based Technologies for Engineering Plant Virus Resistance.},
journal = {Plants (Basel, Switzerland)},
volume = {10},
number = {1},
pages = {},
doi = {10.3390/plants10010082},
pmid = {33401751},
issn = {2223-7747},
support = {14.W03.31.0003//Government of Russian Federation/ ; },
abstract = {In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses.},
}
@article {pmid33293555,
year = {2020},
author = {Carlson-Stevermer, J and Das, A and Abdeen, AA and Fiflis, D and Grindel, BI and Saxena, S and Akcan, T and Alam, T and Kletzien, H and Kohlenberg, L and Goedland, M and Dombroe, MJ and Saha, K},
title = {Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6277},
pmid = {33293555},
issn = {2041-1723},
support = {R35 GM119644/GM/NIGMS NIH HHS/United States ; P30 CA014520/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Computer Simulation ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Therapy/*methods ; Glycogen Storage Disease Type II/genetics/*therapy ; Humans ; Induced Pluripotent Stem Cells ; Infant ; Inheritance Patterns ; Liver/cytology ; Male ; Models, Genetic ; Mutation ; Primary Cell Culture ; },
abstract = {Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.},
}
@article {pmid33229326,
year = {2020},
author = {Lin, MT and Lai, LL and Zhao, M and Lin, BW and Yao, XP},
title = {[Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {10},
pages = {1017-1027},
doi = {10.16288/j.yczz.20-138},
pmid = {33229326},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Mice ; Mice, Knockout ; *Models, Animal ; RNA, Guide/genetics ; *Sodium-Phosphate Cotransporter Proteins, Type III/genetics ; },
abstract = {Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.},
}
@article {pmid33229321,
year = {2020},
author = {Bao, LW and Zhou, YY and Zeng, FY},
title = {[Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {10},
pages = {949-964},
doi = {10.16288/j.yczz.20-110},
pmid = {33229321},
issn = {0253-9772},
mesh = {CRISPR-Cas Systems/genetics ; Clinical Trials as Topic ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; *Genetic Therapy ; *Hemophilia A/genetics/therapy ; Humans ; Research/trends ; Technology ; *beta-Thalassemia/genetics/therapy ; },
abstract = {Thalassemia and hemophilia are common inherited blood disorders caused by genetic abnormalities. These diseases are difficult to cure and can be inherited to the next generation, causing severe family and social burden. The emergence of gene therapy provides a new treatment for genetic diseases. However, since its first clinical trial in 1990, the development of gene therapy has not been as optimistic in the past three decades as one could hope. The development of gene-editing technology, particularly the third generation gene-editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), has given hope in such therapeutic approach for having advantages in high editing efficiency, simple operation, and low cost. Gene editing-mediated gene therapy has thus received increasing attention from the biomedical community. It has shown promises for the treatment of inherited blood disorders, such as thalassemia and hemophilia. This paper reviews the fundamental research progress of gene therapy for β-thalassemia and hemophilia based on CRISPR/Cas9 technology in the past six years. It also summarizes the CRISPR/Cas9-based clinical trials of gene therapy. The problems and possible solutions to this technology for gene therapy are also discussed, thereby providing a reference for the research on gene therapy of inherited blood disorders based on CRISPR/Cas9 technology.},
}
@article {pmid33170892,
year = {2020},
author = {Alghamdi, A and Aldossary, W and Albahkali, S and Alotaibi, B and Alrfaei, BM},
title = {The loss of microglia activities facilitates glaucoma progression in association with CYP1B1 gene mutation (p.Gly61Glu).},
journal = {PloS one},
volume = {15},
number = {11},
pages = {e0241902},
pmid = {33170892},
issn = {1932-6203},
mesh = {Amino Acid Substitution ; Animals ; Apoptosis ; Astrocytes/*cytology/metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Cytochrome P-450 CYP1B1/*genetics ; Cytokines/metabolism ; Glaucoma/*congenital/genetics ; Humans ; Male ; Mesenchymal Stem Cells/*cytology/metabolism ; Microglia/*cytology/metabolism ; Models, Animal ; NADP/metabolism ; *Point Mutation ; Rats ; },
abstract = {BACKGROUND: Glaucoma represents the second main cause of irreversible loss of eyesight worldwide. Progression of the disease is due to changes around the optic nerve, eye structure and optic nerve environment. Focusing on primary congenital glaucoma, which is not completely understood, we report an evaluation of an untested mutation (c.182G>A, p.Gly61Glu) within the CYP1B1 gene in the context of microglia, astrocytes and mesenchymal stem cells. We investigated the behaviours of these cells, which are needed to maintain eye homeostasis, in response to the CYP1B1 mutation.<br><br>METHODS AND RESULTS: CRISPR technology was used to edit normal CYP1B1 genes within normal astrocytes, microglia and stem cells in vitro. Increased metabolic activities were found in microglia and astrocytes 24 hours after CYP1B1 manipulation. However, these activities dropped by 40% after 72 hrs. In addition, the nicotinamide adenine dinucleotide phosphate (NADP)/NADPH reducing equivalent process decreased by 50% on average after 72 hrs of manipulation. The cytokines measured in mutated microglia showed progressive activation leading to apoptosis, which was confirmed with annexin-V. The cytokines evaluated in mutant astrocytes were abnormal in comparison to those in the control.<br><br>CONCLUSIONS: The results suggest a progressive inflammation that was induced by mutations (p.Gly61Glu) on CYP1B1. Furthermore, the mutations enhanced the microglia's loss of activity. We are the first to show the direct impact of the mutation on microglia. This progressive inflammation might be responsible for primary congenital glaucoma complications, which could be avoided via an anti-inflammatory regimen. This finding also reveals that progressive inflammation affects recovery failure after surgeries to relieve glaucoma. Moreover, microglia are important for the survival of ganglion cells, along with the clearing of pathogens and inflammation. The reduction of their activities may jeopardise homeostasis within the optic nerve environment and complicate the protection of optic nerve components (such as retinal ganglion and glial cells).},
}
@article {pmid33147260,
year = {2020},
author = {Liu, H and Robinson, DS and Wu, ZY and Kuo, R and Yoshikuni, Y and Blaby, IK and Cheng, JF},
title = {Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems.},
journal = {PloS one},
volume = {15},
number = {11},
pages = {e0241867},
pmid = {33147260},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genome, Bacterial ; Integrases/*metabolism ; Photorhabdus/*genetics ; },
abstract = {The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.},
}
@article {pmid33137164,
year = {2020},
author = {Schleicher, EM and Dhoonmoon, A and Jackson, LM and Clements, KE and Stump, CL and Nicolae, CM and Moldovan, GL},
title = {Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors.},
journal = {PLoS genetics},
volume = {16},
number = {11},
pages = {e1009176},
pmid = {33137164},
issn = {1553-7404},
support = {R01 ES026184/ES/NIEHS NIH HHS/United States ; R01 GM134681/GM/NIGMS NIH HHS/United States ; F31 CA243301/CA/NCI NIH HHS/United States ; },
mesh = {Apoptosis/drug effects/genetics ; Ataxia Telangiectasia Mutated Proteins/*antagonists &amp; inhibitors ; Biomarkers, Tumor/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; DNA Replication/drug effects/genetics ; Drug Resistance, Neoplasm/*genetics ; Drug Screening Assays, Antitumor ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Mediator Complex/genetics/metabolism ; Neoplasms/*drug therapy/genetics/pathology ; Protein Kinase Inhibitors/*pharmacology/therapeutic use ; Pyrimidines/pharmacology/therapeutic use ; Signal Transduction/drug effects/genetics ; Sulfoxides/pharmacology/therapeutic use ; Transforming Growth Factor beta/metabolism ; },
abstract = {The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFβ signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.},
}
@article {pmid33137136,
year = {2020},
author = {Chen, K and Yu, Y and Yang, D and Yang, X and Tang, L and Liu, Y and Luo, X and R Walter, J and Liu, Z and Xu, J and Huang, Y},
title = {Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori.},
journal = {PLoS genetics},
volume = {16},
number = {11},
pages = {e1009194},
pmid = {33137136},
issn = {1553-7404},
mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*physiology ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/*genetics ; Female ; Gene Expression Regulation, Developmental ; Insect Proteins/genetics/*metabolism ; Male ; Mutation ; Nuclear Proteins/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Sex Determination Processes/*genetics ; },
abstract = {Sex determination pathways are astoundingly diverse in insects. For instance, the silk moth Bombyx mori uniquely use various components of the piRNA pathway to produce the Fem signal for specification of the female fate. In this study, we identified BmGTSF1 as a novel piRNA factor which participates in B. mori sex determination. We found that BmGtsf1 has a distinct expression pattern compared to Drosophila and mouse. CRISPR/Cas9 induced mutation in BmGtsf1 resulted in partial sex reversal in genotypically female animals by shifting expression of the downstream targets BmMasc and Bmdsx to the male pattern. As levels of Fem piRNAs were substantially reduced in female mutants, we concluded that BmGtsf1 plays a critical role in the biogenesis of the feminizing signal. We also demonstrated that BmGTSF1 physically interacted with BmSIWI, a protein previously reported to be involved in female sex determination, indicating BmGTSF1 function as the cofactor of BmSIWI. BmGtsf1 mutation resulted in piRNA pathway dysregulation, including piRNA biogenesis defects and transposon derepression, suggesting BmGtsf1 is also a piRNA factor in the silkworm. Furthermore, we found that BmGtsf1 mutation leads to gametogenesis defects in both male and female. Our data suggested that BmGtsf1 is a new component involved in the sex determination pathway in B. mori.},
}
@article {pmid33087559,
year = {2020},
author = {Lin, RJ and Kuo, MW and Yang, BC and Tsai, HH and Chen, K and Huang, JR and Lee, YS and Yu, AL and Yu, J},
title = {B3GALT5 knockout alters gycosphingolipid profile and facilitates transition to human naïve pluripotency.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {44},
pages = {27435-27444},
doi = {10.1073/pnas.2003155117},
pmid = {33087559},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems/genetics ; *Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; Galactosyltransferases/genetics/*metabolism ; Gene Knockdown Techniques ; Glycosphingolipids/*metabolism ; Humans ; Pluripotent Stem Cells/*metabolism ; Stage-Specific Embryonic Antigens/*metabolism ; },
abstract = {Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of β-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca2+, and conducive for transition to naïve pluripotency.},
}
@article {pmid32865499,
year = {2020},
author = {Müller, M and Schneider, M and Salathé, M and Vayena, E},
title = {Assessing Public Opinion on CRISPR-Cas9: Combining Crowdsourcing and Deep Learning.},
journal = {Journal of medical Internet research},
volume = {22},
number = {8},
pages = {e17830},
pmid = {32865499},
issn = {1438-8871},
mesh = {CRISPR-Cas Systems/*physiology ; Crowdsourcing/*methods ; Deep Learning/*standards ; Humans ; *Public Opinion ; },
abstract = {BACKGROUND: The discovery of the CRISPR-Cas9-based gene editing method has opened unprecedented new potential for biological and medical engineering, sparking a growing public debate on both the potential and dangers of CRISPR applications. Given the speed of technology development and the almost instantaneous global spread of news, it is important to follow evolving debates without much delay and in sufficient detail, as certain events may have a major long-term impact on public opinion and later influence policy decisions.<br><br>OBJECTIVE: Social media networks such as Twitter have shown to be major drivers of news dissemination and public discourse. They provide a vast amount of semistructured data in almost real-time and give direct access to the content of the conversations. We can now mine and analyze such data quickly because of recent developments in machine learning and natural language processing.<br><br>METHODS: Here, we used Bidirectional Encoder Representations from Transformers (BERT), an attention-based transformer model, in combination with statistical methods to analyze the entirety of all tweets ever published on CRISPR since the publication of the first gene editing application in 2013.<br><br>RESULTS: We show that the mean sentiment of tweets was initially very positive, but began to decrease over time, and that this decline was driven by rare peaks of strong negative sentiments. Due to the high temporal resolution of the data, we were able to associate these peaks with specific events and to observe how trending topics changed over time.<br><br>CONCLUSIONS: Overall, this type of analysis can provide valuable and complementary insights into ongoing public debates, extending the traditional empirical bioethics toolset.},
}
@article {pmid32811847,
year = {2020},
author = {Yokouchi, Y and Suzuki, S and Ohtsuki, N and Yamamoto, K and Noguchi, S and Soejima, Y and Goto, M and Ishioka, K and Nakamura, I and Suzuki, S and Takenoshita, S and Era, T},
title = {Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13927},
pmid = {32811847},
issn = {2045-2322},
mesh = {Alleles ; Bacterial Proteins/genetics/*metabolism ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Collagen Type VII/genetics/metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Endonucleases/genetics ; Gene Editing/*methods ; Genome, Human/genetics ; Humans ; Induced Pluripotent Stem Cells/physiology ; Mutation/genetics ; Nucleotides/genetics ; Pluripotent Stem Cells/physiology ; Polymorphism, Single Nucleotide/*genetics ; Proto-Oncogene Proteins c-ret/genetics/metabolism ; },
abstract = {Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.},
}
@article {pmid32745724,
year = {2020},
author = {D'Amore, C and Moro, E and Borgo, C and Itami, K and Hirota, T and Pinna, LA and Salvi, M},
title = {&quot;Janus&quot; efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanisms.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1867},
number = {11},
pages = {118807},
doi = {10.1016/j.bbamcr.2020.118807},
pmid = {32745724},
issn = {1879-2596},
mesh = {CRISPR-Cas Systems/genetics ; Casein Kinase II/antagonists &amp; inhibitors/*genetics ; Cell Death/drug effects/*genetics ; Gene Editing ; Hep G2 Cells ; Humans ; Indoles/pharmacology ; Neoplasms/*drug therapy ; Pinocytosis/drug effects/genetics ; Pyrimidines/pharmacology ; Quinolines/pharmacology ; Vacuoles/drug effects/genetics ; rac1 GTP-Binding Protein/antagonists &amp; inhibitors/*genetics ; },
abstract = {Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.},
}
@article {pmid32658961,
year = {2020},
author = {Domènech, EB and Andrés, R and López-Iniesta, MJ and Mirra, S and García-Arroyo, R and Milla, S and Sava, F and Andilla, J and Loza-Álvarez, P and de la Villa, P and Gonzàlez-Duarte, R and Marfany, G},
title = {A New Cerkl Mouse Model Generated by CRISPR-Cas9 Shows Progressive Retinal Degeneration and Altered Morphological and Electrophysiological Phenotype.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {61},
number = {8},
pages = {14},
pmid = {32658961},
issn = {1552-5783},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; DNA/*genetics ; DNA Mutational Analysis ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; *Mutation ; Phenotype ; Phosphotransferases (Alcohol Group Acceptor)/*genetics/metabolism ; Retinal Cone Photoreceptor Cells/*metabolism/pathology ; Retinal Degeneration/*genetics/metabolism/pathology ; Retinal Pigment Epithelium/*metabolism/pathology ; },
abstract = {Purpose: Close to 100 genes cause retinitis pigmentosa, a Mendelian rare disease that affects 1 out of 4000 people worldwide. Mutations in the ceramide kinase-like gene (CERKL) are a prevalent cause of autosomal recessive cause retinitis pigmentosa and cone-rod dystrophy, but the functional role of this gene in the retina has yet to be fully determined. We aimed to generate a mouse model that resembles the phenotypic traits of patients carrying CERKL mutations to undertake functional studies and assay therapeutic approaches.<br><br>Methods: The Cerkl locus has been deleted (around 97 kb of genomic DNA) by gene editing using the CRISPR-Cas9 D10A nickase. Because the deletion of the Cerkl locus is lethal in mice in homozygosis, a double heterozygote mouse model with less than 10% residual Cerkl expression has been generated. The phenotypic alterations of the retina of this new model have been characterized at the morphological and electrophysiological levels.<br><br>Results: This CerklKD/KO model shows retinal degeneration, with a decreased number of cones and progressive photoreceptor loss, poorly stacked photoreceptor outer segment membranes, defective retinal pigment epithelium phagocytosis, and altered electrophysiological recordings in aged retinas.<br><br>Conclusions: To our knowledge, this is the first Cerkl mouse model to mimic many of the phenotypic traits, including the slow but progressive retinal degeneration, shown by human patients carrying CERKL mutations. This useful model will provide unprecedented insights into the retinal molecular pathways altered in these patients and will contribute to the design of effective treatments.},
}
@article {pmid32422169,
year = {2020},
author = {Przewrocka, J and Rowan, A and Rosenthal, R and Kanu, N and Swanton, C},
title = {Unintended on-target chromosomal instability following CRISPR/Cas9 single gene targeting.},
journal = {Annals of oncology : official journal of the European Society for Medical Oncology},
volume = {31},
number = {9},
pages = {1270-1273},
pmid = {32422169},
issn = {1569-8041},
support = {FC001169/MRC_/Medical Research Council/United Kingdom ; FC001169/WT_/Wellcome Trust/United Kingdom ; FC001169/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Chromosomal Instability ; *Gene Targeting ; Humans ; },
}
@article {pmid32394708,
year = {2020},
author = {Xie, T and Chen, X and Guo, T and Rong, H and Chen, Z and Sun, Q and Batley, J and Jiang, J and Wang, Y},
title = {Targeted Knockout of BnTT2 Homologues for Yellow-Seeded Brassica napus with Reduced Flavonoids and Improved Fatty Acid Composition.},
journal = {Journal of agricultural and food chemistry},
volume = {68},
number = {20},
pages = {5676-5690},
doi = {10.1021/acs.jafc.0c01126},
pmid = {32394708},
issn = {1520-5118},
mesh = {Brassica napus/chemistry/*genetics/metabolism ; CRISPR-Cas Systems ; Color ; Fatty Acids/*chemistry/metabolism ; Flavonoids/*chemistry/metabolism ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified/chemistry/genetics/metabolism ; Seeds/chemistry/genetics/*metabolism ; },
abstract = {Brassica napus is one of the important oil crops grown worldwide, and oil quality improvement is a major goal in rapeseed breeding. Yellow seed is an excellent trait, which has great potential in improving seed quality and economic value. In this study, we created stable yellow seed mutants using a CRISPR/Cas9 system and obtained the yellow seed phenotype only when the four alleles of two BnTT2 homologues were knocked out, indicating that the two BnTT2 homologues had conserved but redundant functions in regulating seed color. Histochemical staining and flavonoid metabolic analysis proved that the BnTT2 mutation hindered the synthesis and accumulation of proanthocyanidins. Transcriptome analysis also showed that the BnTT2 mutation inhibited the expression of genes in the phenylpropanoid and flavonoid biosynthetic pathway, which might be regulated by the complex of BnTT2, BnTT8 and BnTTG1. In addition, the homozygous mutants of BnTT2 homologues increased oil content and improved fatty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3), which could be used for the genetic improvement of rapeseed. Overall, this research showed that the BnTT2 mutation can be used for yellow seed breeding and oil improvement, which is of great significance in improving the economic value of rapeseeds.},
}
@article {pmid32372640,
year = {2020},
author = {Shu, P and Li, Z and Min, D and Zhang, X and Ai, W and Li, J and Zhou, J and Li, Z and Li, F and Li, X},
title = {CRISPR/Cas9-Mediated SlMYC2 Mutagenesis Adverse to Tomato Plant Growth and MeJA-Induced Fruit Resistance to Botrytis cinerea.},
journal = {Journal of agricultural and food chemistry},
volume = {68},
number = {20},
pages = {5529-5538},
doi = {10.1021/acs.jafc.9b08069},
pmid = {32372640},
issn = {1520-5118},
mesh = {Acetates/*adverse effects/pharmacology ; Botrytis/*physiology ; CRISPR-Cas Systems ; Cyclopentanes/*adverse effects/pharmacology ; Disease Resistance ; Flowers/genetics/growth &amp; development/immunology ; Fruit/drug effects/genetics/immunology/microbiology ; Gene Expression Regulation, Plant ; Lycopersicon esculentum/drug effects/*genetics/immunology/microbiology ; Mutagenesis ; Oxylipins/*adverse effects/pharmacology ; Plant Diseases/immunology/*microbiology ; Plant Growth Regulators/*pharmacology ; Plant Proteins/*genetics/immunology ; Plants, Genetically Modified/genetics/growth &amp; development/*immunology/microbiology ; },
abstract = {Methyl jasmonate (MeJA), a natural phytohormone, played a critical role not only in plant growth but also in plant defense response to biotic and abiotic stresses. MYC2, a basic helix-loop-helix transcription factor, is a master regulator in MeJA signaling pathway. In the present work, slmyc2 mutants were generated by the clustered regularly interspaced short palindromic repeats and associated Cas9 protein (CRISPR/Cas9) system to investigate the role of SlMYC2 in tomato plant growth and fruit disease resistance induced by exogenous MeJA. The results showed that slmyc2 mutants possessed a higher number of flowers and a lower fruit setting rate in comparison with wild-type plants. In addition, the fruit shape of slmyc2 mutant was prolate, while the control fruits were oblate. Knockout of SlMYC2 significantly decreased the activities of disease defensive and antioxidant enzymes, as well as the expression levels of pathogen-related (PR) genes (SlPR-1 and SlPR-STH2) and the key genes related to jasmonic acid (JA) biosynthesis and signaling pathway including allene oxide cyclase (SlAOC), lipoxygenase D (SlLOXD), SlMYC2, and coronatine insensitive 1 (SlCOI1), and consequently aggravated the disease symptoms. By contrast, the disease symptoms were largely reduced in MeJA-treated fruit that possessed higher activities of these enzymes and expression levels of genes. However, the induction effects of MeJA on fruit disease resistance and these enzymes' activities and genes' expressions were significantly attenuated by knockout of SlMYC2. Therefore, the results indicated that SlMYC2 played positive regulatory roles not only in the growth of tomato plants but also in MeJA-induced disease resistance and the antioxidant process in tomato fruits.},
}
@article {pmid32366884,
year = {2020},
author = {Pinzon-Arteaga, C and Snyder, MD and Lazzarotto, CR and Moreno, NF and Juras, R and Raudsepp, T and Golding, MC and Varner, DD and Long, CR},
title = {Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {7411},
pmid = {32366884},
issn = {2045-2322},
mesh = {Animals ; Apoptosis ; Biotechnology/methods ; *CRISPR-Cas Systems ; Cell Line ; *Exons ; Fibroblasts/*metabolism ; *Gene Editing ; Genetic Engineering/methods ; Glycogen Storage Disease Type IV/*genetics/therapy/veterinary ; Homologous Recombination ; Horses ; Karyotyping ; Phenotype ; *Point Mutation ; RNA, Guide/genetics ; Skin/metabolism ; },
abstract = {Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1102C>A). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1102C>A mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.},
}
@article {pmid32355272,
year = {2020},
author = {Audebert, C and Bonardi, F and Caboche, S and Guyot, K and Touzet, H and Merlin, S and Gantois, N and Creusy, C and Meloni, D and Mouray, A and Viscogliosi, E and Certad, G and Benamrouz-Vanneste, S and Chabé, M},
title = {Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {7316},
pmid = {32355272},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Computational Biology ; Cryptosporidiosis/*parasitology ; Cryptosporidium parvum/*genetics/pathogenicity ; Feces ; Female ; Genome ; Genome, Protozoan ; Humans ; Male ; Mice ; Mice, SCID ; Middle Aged ; Oocysts ; Phenotype ; Virulence/*genetics ; Virulence Factors/*genetics ; Young Adult ; },
abstract = {Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.},
}
@article {pmid32346083,
year = {2020},
author = {Soliman, SHA and Stark, AE and Gardner, ML and Harshman, SW and Breece, CC and Amari, F and Orlacchio, A and Chen, M and Tessari, A and Martin, JA and Visone, R and Freitas, MA and La Perle, KMD and Palmieri, D and Coppola, V},
title = {Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {7138},
pmid = {32346083},
issn = {2045-2322},
support = {P30 CA016058/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Cytoskeletal Proteins/genetics/*metabolism ; Immunohistochemistry ; Mice ; Mice, Knockout ; Nuclear Proteins/genetics/*metabolism ; Phosphoproteins/*metabolism ; Protein Binding ; RNA, Messenger/metabolism ; RNA-Binding Proteins/*metabolism ; },
abstract = {The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.},
}
@article {pmid32300998,
year = {2020},
author = {Wu, C and Chen, Y and Qiu, Y and Niu, X and Zhu, N and Chen, J and Yao, H and Wang, W and Ma, Y},
title = {A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription.},
journal = {Biotechnology letters},
volume = {42},
number = {7},
pages = {1203-1210},
doi = {10.1007/s10529-020-02887-0},
pmid = {32300998},
issn = {1573-6776},
support = {2019M661402//Postdoctoral Research Foundation of China/ ; 222201714053//the Fundamental Research Funds for the Central Universities/ ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Fungal/*genetics ; Hypocreales/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; Transcription, Genetic/genetics ; },
abstract = {OBJECTIVE: To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription.<br><br>RESULTS: Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500 bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events.<br><br>CONCLUSIONS: We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.},
}
@article {pmid32295026,
year = {2020},
author = {Anwar, A and Kim, JK},
title = {Transgenic Breeding Approaches for Improving Abiotic Stress Tolerance: Recent Progress and Future Perspectives.},
journal = {International journal of molecular sciences},
volume = {21},
number = {8},
pages = {},
pmid = {32295026},
issn = {1422-0067},
support = {PJ013666//Rural Development Administration/ ; NRF-2017R1A2B4007457//National Research Foundation of Korea, Ministry of Education/ ; },
mesh = {*Adaptation, Biological ; Agriculture ; CRISPR-Cas Systems ; Crops, Agricultural/*genetics/*metabolism ; Gene Editing ; Gene Expression Regulation, Plant ; Genetic Engineering ; MicroRNAs ; *Plant Breeding ; *Plants, Genetically Modified ; Quantitative Trait Loci ; *Stress, Physiological/genetics ; },
abstract = {The recent rapid climate changes and increasing global population have led to an increased incidence of abiotic stress and decreased crop productivity. Environmental stresses, such as temperature, drought, nutrient deficiency, salinity, and heavy metal stresses, are major challenges for agriculture, and they lead to a significant reduction in crop growth and productivity. Abiotic stress is a very complex phenomenon, involving a variety of physiological and biochemical changes in plant cells. Plants exposed to abiotic stress exhibit enhanced levels of ROS (reactive oxygen species), which are highly reactive and toxic and affect the biosynthesis of chlorophyll, photosynthetic capacity, and carbohydrate, protein, lipid, and antioxidant enzyme activities. Transgenic breeding offers a suitable alternative to conventional breeding to achieve plant genetic improvements. Over the last two decades, genetic engineering/transgenic breeding techniques demonstrated remarkable developments in manipulations of the genes for the induction of desired characteristics into transgenic plants. Transgenic approaches provide us with access to identify the candidate genes, miRNAs, and transcription factors (TFs) that are involved in specific plant processes, thus enabling an integrated knowledge of the molecular and physiological mechanisms influencing the plant tolerance and productivity. The accuracy and precision of this phenomenon assures great success in the future of plant improvements. Hence, transgenic breeding has proven to be a promising tool for abiotic stress improvement in crops. This review focuses on the potential and successful applications, recent progress, and future perspectives of transgenic breeding for improving abiotic stress tolerance and productivity in plants.},
}
@article {pmid32198422,
year = {2020},
author = {Hanzawa, N and Hashimoto, K and Yuan, X and Kawahori, K and Tsujimoto, K and Hamaguchi, M and Tanaka, T and Nagaoka, Y and Nishina, H and Morita, S and Hatada, I and Yamada, T and Ogawa, Y},
title = {Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {5181},
pmid = {32198422},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Demethylation ; DNA Methylation ; Epigenesis, Genetic ; Epigenome ; Fibroblast Growth Factors/*genetics/metabolism ; Gene Editing/methods ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; PPAR alpha/genetics/metabolism ; Promoter Regions, Genetic/genetics ; },
abstract = {Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.},
}
@article {pmid32192326,
year = {2020},
author = {Tu, K and Deng, H and Kong, L and Wang, Y and Yang, T and Hu, Q and Hu, M and Yang, C and Zhang, Z},
title = {Reshaping Tumor Immune Microenvironment through Acidity-Responsive Nanoparticles Featured with CRISPR/Cas9-Mediated Programmed Death-Ligand 1 Attenuation and Chemotherapeutics-Induced Immunogenic Cell Death.},
journal = {ACS applied materials &amp; interfaces},
volume = {12},
number = {14},
pages = {16018-16030},
doi = {10.1021/acsami.9b23084},
pmid = {32192326},
issn = {1944-8252},
mesh = {Acids/chemistry ; Animals ; B7-H1 Antigen/genetics/immunology ; CRISPR-Cas Systems/genetics/immunology ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Cyclin-Dependent Kinase 5/genetics ; Humans ; Immunogenic Cell Death/drug effects/genetics ; Melanoma, Experimental/genetics/*immunology/pathology/therapy ; Mice ; Nanoparticles/*chemistry/therapeutic use ; Paclitaxel/chemistry/immunology/*pharmacology ; T-Lymphocytes/drug effects/immunology ; Transfection ; Tumor Microenvironment/drug effects ; },
abstract = {Blocking immune checkpoints with monoclonal antibody has been verified to achieve potential clinical successes for cancer immunotherapy. However, its application has been impeded by the &quot;cold&quot; tumor microenvironment. Here, weak acidity-responsive nanoparticles co-loaded with CRISPR/Cas9 and paclitaxel (PTX) with the ability to convert &quot;cold&quot; tumor into &quot;hot&quot; tumor are reported. The nanoparticles exhibited high cargo packaging capacity, superior transfection efficiency, well biocompatibility, and effective tumor accumulation. The CRISPR/Cas9 encapsulated in nanoparticles could specifically knock out cyclin-dependent kinase 5 gene to significantly attenuate the expression of programmed death-ligand 1 on tumor cells. More importantly, PTX co-delivered in nanoparticles could significantly induce immunogenic cell death, reduce regulatory T lymphocytes, repolarize tumor-associated macrophages, and enhance antitumor immunity. Therefore, the nanoparticles could effectively convert cold tumor into hot tumor, achieve effective tumor growth inhibition, and prolong overall survival from 16 to 36 days. This research provided a referable strategy for the development of combinatorial immunotherapy and chemotherapy.},
}
@article {pmid32090297,
year = {2020},
author = {Zhang, XK and Wang, DN and Chen, J and Liu, ZJ and Wei, LJ and Hua, Q},
title = {Metabolic engineering of β-carotene biosynthesis in Yarrowia lipolytica.},
journal = {Biotechnology letters},
volume = {42},
number = {6},
pages = {945-956},
doi = {10.1007/s10529-020-02844-x},
pmid = {32090297},
issn = {1573-6776},
support = {21576089, 21776081//National Natural Science Foundation of China/ ; B18022//the 111 Project and Research Program of State Key Laboratory of Bioreactor Engineering./ ; },
mesh = {Bioreactors ; CRISPR-Cas Systems/genetics ; Fermentation ; Glucose/metabolism ; Metabolic Engineering/*methods ; Yarrowia/*genetics/metabolism ; beta Carotene/analysis/*metabolism ; },
abstract = {OBJECTIVE: Carotenoids, as potent antioxidant compounds, have gained extensive attention, especially in human health. In this study, the combination of CRISPR/Cas9 integration strategy and fermenter cultivation was utilized to obtain efficient β-carotene-producing Yarrowia lipolytica cell factories for potential industrial application.<br><br>RESULTS: The introduction of the genes of Mucor circinelloides, encoding phytoene dehydrogenase (carB) and bifunctional phytoene synthase/lycopene cyclase (carRP), contributed to the heterologous production of β-carotene in Y. lipolytica XK2. Furthermore, β-carotene production was efficiently enhanced by increasing the copy numbers of the carB and carRP genes and overexpressing of GGS1, ERG13, and HMG, the genes related to the mevalonate (MVA) pathway. Thus, the optimized strain overexpressed a total of eight genes, including three copies of carRP, two copies of carB, and single copies of GGS1, HMG, and ERG13. As a consequence, strain Y. lipolytica XK19 accumulated approximately 408 mg/L β-carotene in shake flask cultures, a twenty-four-fold increase compared to the parental strain Y. lipolytica XK2.<br><br>CONCLUSIONS: 4.5 g/L β-carotene was obtained in a 5-L fermenter through a combination of genetic engineering and culture optimization, suggesting a great capacity and flexibility of Y. lipolytica in the production of carotenoids.},
}
@article {pmid31953182,
year = {2020},
author = {Rojas-Sánchez, U and López-Calleja, AC and Millán-Chiu, BE and Fernández, F and Loske, AM and Gómez-Lim, MA},
title = {Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9.},
journal = {Protein expression and purification},
volume = {168},
number = {},
pages = {105570},
doi = {10.1016/j.pep.2020.105570},
pmid = {31953182},
issn = {1096-0279},
mesh = {Aspergillus niger/*genetics/metabolism ; CRISPR-Cas Systems ; Cloning, Molecular ; Erythropoietin/*biosynthesis/genetics ; Fructose-Bisphosphatase/chemistry/genetics ; Gene Expression ; Gene Knockdown Techniques ; *Genes, Fungal ; Genetic Vectors/chemistry/metabolism ; Glycosylation ; Humans ; *Introns ; Plasmids/chemistry/*metabolism ; Promoter Regions, Genetic ; Protein Stability ; Proteolysis ; RNA, Messenger/*genetics/metabolism ; Recombinant Proteins/biosynthesis/genetics ; },
abstract = {Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L-1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.},
}
@article {pmid31914394,
year = {2020},
author = {Kondo, S and Takahashi, T and Yamagata, N and Imanishi, Y and Katow, H and Hiramatsu, S and Lynn, K and Abe, A and Kumaraswamy, A and Tanimoto, H},
title = {Neurochemical Organization of the Drosophila Brain Visualized by Endogenously Tagged Neurotransmitter Receptors.},
journal = {Cell reports},
volume = {30},
number = {1},
pages = {284-297.e5},
doi = {10.1016/j.celrep.2019.12.018},
pmid = {31914394},
issn = {2211-1247},
mesh = {Animals ; Brain/*diagnostic imaging ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/genetics/*metabolism ; Ethanol/adverse effects ; Gene Expression Regulation ; Genes, Reporter ; Receptors, Dopamine/metabolism ; Receptors, Neurotransmitter/*metabolism ; },
abstract = {Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. Here we describe a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, we profile the expression of 75 neurotransmitter receptors in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, we convert T2A-GAL4 into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. Our resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain.},
}
@article {pmid31827259,
year = {2020},
author = {Schuh, RS and Gonzalez, EA and Tavares, AMV and Seolin, BG and Elias, LS and Vera, LNP and Kubaski, F and Poletto, E and Giugliani, R and Teixeira, HF and Matte, U and Baldo, G},
title = {Neonatal nonviral gene editing with the CRISPR/Cas9 system improves some cardiovascular, respiratory, and bone disease features of the mucopolysaccharidosis I phenotype in mice.},
journal = {Gene therapy},
volume = {27},
number = {1-2},
pages = {74-84},
pmid = {31827259},
issn = {1476-5462},
mesh = {Animals ; Animals, Newborn ; Bone Diseases/genetics ; CRISPR-Cas Systems/genetics ; Cardiovascular System/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; Female ; Gene Editing/methods ; Genetic Therapy/methods ; Glycosaminoglycans/metabolism ; Iduronidase/*genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mucopolysaccharidosis I/genetics/physiopathology/*therapy ; Phenotype ; RNA, Untranslated/*genetics/metabolism ; },
abstract = {Mucopolysaccharidosis type I (MPS I) is caused by deficiency of alpha-L-iduronidase (IDUA), leading to multisystemic accumulation of glycosaminoglycans (GAG). Untreated MPS I patients may die in the first decades of life, mostly due to cardiovascular and respiratory complications. We previously reported that the treatment of newborn MPS I mice with intravenous administration of lipossomal CRISPR/Cas9 complexes carrying the murine Idua gene aiming at the ROSA26 locus resulted in long-lasting IDUA activity and GAG reduction in various tissues. Following this, the present study reports the effects of gene editing in cardiovascular, respiratory, bone, and neurologic functions in MPS I mice. Bone morphology, specifically the width of zygomatic and femoral bones, showed partial improvement. Although heart valves were still thickened, cardiac mass and aortic elastin breaks were reduced, with normalization of aortic diameter. Pulmonary resistance was normalized, suggesting improvement in respiratory function. In contrast, behavioral abnormalities and neuroinflammation still persisted, suggesting deterioration of the neurological functions. The set of results shows that gene editing performed in newborn animals improved some manifestations of the MPS I disorder in bone, respiratory, and cardiovascular systems. However, further studies will be imperative to find better delivery strategies to reach &quot;hard-to-treat&quot; tissues to ensure better systemic and neurological effects.},
}
@article {pmid31776930,
year = {2020},
author = {Zhang, J and Späth, SS and Katz, SG},
title = {Genome-Wide CRISPRi/a Screening in an In Vitro Coculture Assay of Human Immune Cells with Tumor Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2097},
number = {},
pages = {231-252},
pmid = {31776930},
issn = {1940-6029},
support = {R01 HL131793/HL/NHLBI NIH HHS/United States ; R21 AI121993/AI/NIAID NIH HHS/United States ; R21 CA198561/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Coculture Techniques/*methods ; *Genetic Testing ; *Genome ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Lentivirus/metabolism ; Leukocytes/*immunology ; Neoplasms/*pathology ; Oligonucleotides/metabolism ; Plasmids/metabolism ; RNA, Guide/metabolism ; Transformation, Genetic ; Virion/metabolism ; },
abstract = {Cell-based immunotherapy has achieved preclinical success in certain types of cancer patients, with a few approved cell-based products for clinical use. These achievements revitalized the field of cell engineering/ immunotherapy and brought attention to the opportunities that cell-based immunotherapeutics can offer to patients. On the other hand, obvious indications emphasize the need for a better understanding of the biological mechanisms involved in the immune response. This knowledge may not only ameliorate safety and efficacy, but also determine the possibilities and limitations in use of immune cell engineering for cancer treatment, and facilitate developing novel immunotherapeutic strategies. Recently developed technology based on CRISPR-dCas9 has an immense potential to systematically uncover genetic mechanisms by identifying subsets of essential genes involved in interactions of cancer cells with the immune system. This chapter will present a reliable and reproducible general protocol for the application of genome-wide sgRNA gene-editing tools in the recently established two-cell type co-culture, consisting of immune cells as effectors and cancer cells as targets, utilizing CRISPRi/a-dCas9-based technology.},
}
@article {pmid31776929,
year = {2020},
author = {Bailis, W},
title = {CRISPR/Cas9 Gene Targeting in Primary Mouse Bone Marrow-Derived Macrophages.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2097},
number = {},
pages = {223-230},
doi = {10.1007/978-1-0716-0203-4_14},
pmid = {31776929},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Gene Targeting/*methods ; HEK293 Cells ; Humans ; Macrophages/*metabolism ; Mice ; Retroviridae/metabolism ; Transduction, Genetic ; },
abstract = {CRISPR-Cas9 technology allows for rapid, targeted genome editing at nearly any loci with limited off-target effects. Here, we describe a method for using retroviral transduction to deliver single-guide RNA to primary bone marrow-derived macrophages. This protocol allows for high-throughput reverse genetics assays in primary immune cells and is also compatible with retroviral systems for transgene expression.},
}
@article {pmid31702097,
year = {2020},
author = {Niu, Q and Wu, S and Li, Y and Yang, X and Liu, P and Xu, Y and Lang, Z},
title = {Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.},
journal = {Journal of integrative plant biology},
volume = {62},
number = {4},
pages = {398-402},
doi = {10.1111/jipb.12886},
pmid = {31702097},
issn = {1744-7909},
support = {2018YFD1000200//National Key Research and Development Program/ ; 2019ZX08010003-002-006//Major Project of Transgenic Crops of China/ ; XDB27040000//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genome, Plant ; Lycopersicon esculentum/*genetics ; Protoplasts/metabolism ; },
abstract = {The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome-editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9-NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9-NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG-Cas9) based on mutations from both xCas9 and Cas9-NG, and found that XNG-Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG-Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9-NG) active in plant.},
}
@article {pmid31570788,
year = {2020},
author = {Nicolai, S and Mahen, R and Raschellà, G and Marini, A and Pieraccioli, M and Malewicz, M and Venkitaraman, AR and Melino, G},
title = {ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining.},
journal = {Oncogene},
volume = {39},
number = {4},
pages = {754-766},
doi = {10.1038/s41388-019-1028-7},
pmid = {31570788},
issn = {1476-5594},
support = {MC_U132670600/MRC_/Medical Research Council/United Kingdom ; MC_UU_00025/2/MRC_/Medical Research Council/United Kingdom ; 100090/12/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; DNA-Binding Proteins/metabolism ; Databases, Genetic ; Humans ; Neoplasms/*genetics/metabolism/*pathology ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; Prognosis ; Repressor Proteins/genetics/*metabolism ; Survival Rate ; },
abstract = {Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients' stratification and for the development of personalised therapeutic strategies.},
}
@article {pmid31548614,
year = {2020},
author = {Ko, T and Sharma, R and Li, S},
title = {Genome-wide screening identifies novel genes implicated in cellular sensitivity to BRAFV600E expression.},
journal = {Oncogene},
volume = {39},
number = {4},
pages = {723-738},
doi = {10.1038/s41388-019-1022-0},
pmid = {31548614},
issn = {1476-5594},
mesh = {3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)/genetics/*metabolism ; Apoptosis ; CRISPR-Cas Systems ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Fibroblasts/metabolism ; *Genome, Human ; Humans ; MAP Kinase Signaling System ; Melanocytes/metabolism ; Mitogen-Activated Protein Kinase 1/genetics/metabolism ; *Mutation ; Neoplasms/genetics/metabolism/*pathology ; Proto-Oncogene Proteins B-raf/*genetics/*metabolism ; Receptors, G-Protein-Coupled/genetics/*metabolism ; Signal Transduction ; },
abstract = {The V600E mutation of BRAF (BRAFV600E), which constitutively activates the ERK/MAPK signaling pathway, is frequently found in melanoma and other cancers. Like most other oncogenes, BRAFV600E causes oncogenic stress to normal cells, leading to growth arrest (senescence) or apoptosis. Through genome-wide screening, we identified genes implicated in sensitivity of human skin melanocytes and fibroblasts to BRAFV600E overexpression. Among the identified genes shared by the two cell types are proto-oncogenes ERK2, a component of the ERK/MAPK pathway, and VAV1, a guanine nucleotide exchange factor for Rho family GTPases that also activates the ERK/MAPK pathway. CDKN1A, which has been known to promote senescence of fibroblasts but not melanocytes, is implicated in sensitivity of the fibroblasts but not the melanocytes to BRAFV600E overexpression. Disruptions of GPR4, a pH-sensing G-protein coupled receptor, and DBT, a subunit of the branched chain α-keto acid dehydrogenase that is required for the second and rate-limiting step of branched amino acid catabolism and implicated in maple syrup urine disease, are the most highly selected in the melanocytes upon BRAFV600E overexpression. Disruption of DBT severely attenuates ERK/MAPK signaling, p53 activation, and apoptosis in melanocytes, at least in part due to accumulation of branched chain α-keto acids. The expression level of BRAF positively correlates with that of DBT in all cancer types and with that of GPR4 in most cancer types. Overexpression of DBT kills all four melanoma cell lines tested regardless of the presence of BRAFV600E mutation. Our findings shed new lights on regulations of oncogenic stress signaling and may be informative for development of novel cancer treatment strategies.},
}
@article {pmid33401227,
year = {2021},
author = {Pan, C and Sretenovic, S and Qi, Y},
title = {CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants.},
journal = {Current opinion in plant biology},
volume = {60},
number = {},
pages = {101980},
doi = {10.1016/j.pbi.2020.101980},
pmid = {33401227},
issn = {1879-0356},
abstract = {The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated) system-mediated precise genome editing has revolutionized genome engineering due to ease of use and versatility of multiplexing. Catalytically inactivated Cas variants (dCas) further expand the usefulness of the CRISPR/Cas system for genetics studies and translational research without inducing DNA double-strand breaks. Fusion of diverse effector domains to dCas proteins empowers the CRISPR/dCas system as a multifunctional platform for gene expression regulation, epigenetic regulation and sequence-specific imaging. In this short review, we summarize the recent advances of CRISPR/dCas-mediated transcriptional activation and repression, and epigenetic modifications. We also highlight the future directions and broader applications of the CRISPR/dCas systems in plants.},
}
@article {pmid33399467,
year = {2021},
author = {Qin, Z and Yang, Y and Yu, S and Liu, L and Chen, Y and Chen, J and Zhou, J},
title = {Repurposing the Endogenous Type I-E CRISPR/Cas System for Gene Repression in Gluconobacter oxydans WSH-003.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.0c00456},
pmid = {33399467},
issn = {2161-5063},
abstract = {Gluconobacter oxydans is well-known for its incomplete oxidizing capacity and has been widely applied in industrial production. However, genetic tools in G. oxydans are still scarce compared with model microorganisms, limiting its metabolic engineering. This study aimed to develop a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system based on the typical type I-E endogenous CRISPR/CRISPR-associated proteins (Cas) system in G. oxydans WSH-003. The nuclease Cas3 in this system was inactivated naturally and hence did not need to be knocked out. Subsequently, the CRISPRi effect was verified by repressing the expression of fluorescent proteins, revealing effective multiplex gene repression. Finally, the endogenous CRISPRi system was used to study the role of the central carbon metabolism pathway, including the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP), in G. oxydans WSH-003. This was done to demonstrate a metabolic engineering application. The PPP was found to be important for cell growth and the substrate conversion rate. The development of the CRISPRi system enriched the gene regulation tools in G. oxydans and promoted the metabolic engineering modification of G. oxydans to improve its performance. In addition, it might have implications for metabolic engineering modification of other genetically recalcitrant strains.},
}
@article {pmid33398095,
year = {2021},
author = {Smith, LM and Jackson, SA and Malone, LM and Ussher, JE and Gardner, PP and Fineran, PC},
title = {The Rcs stress response inversely controls surface and CRISPR-Cas adaptive immunity to discriminate plasmids and phages.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {33398095},
issn = {2058-5276},
abstract = {Bacteria harbour multiple innate defences and adaptive CRISPR-Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR-Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR-Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR-Cas systems. Rcs-mediated repression of CRISPR-Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.},
}
@article {pmid33397707,
year = {2021},
author = {Goh, YJ and Barrangou, R},
title = {A portable CRISPR-Cas9N system for flexible genome engineering in Lactobacillus acidophilus, Lactobacillus gasseri and Lactobacillus paracasei.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1128/AEM.02669-20},
pmid = {33397707},
issn = {1098-5336},
abstract = {Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplements industries, and specific strains such as Lactobacillus acidophilus NCFM have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9N under the regulation of a Lactobacillus promoter and a cloning region for customized sgRNA and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS and ltaS), yielding 35-100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted a mCherry fluorescent protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate concurrent single base substitution and gene deletion in Lactobacillus gasseri ATCC 33323, and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli, and open new avenues for the engineering of health-promoting lactic acid bacteria.Importance This work describes the development of a broad-host range CRISPR-based editing system for genome manipulations in three Lactobacillus species, which belong to lactic acid bacteria (LAB) commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiota, and their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus, and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems, and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.},
}
@article {pmid33397704,
year = {2021},
author = {Synefiaridou, D and Veening, JW},
title = {Harnessing CRISPR-Cas9 for genome editing in Streptococcus pneumoniae D39V.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1128/AEM.02762-20},
pmid = {33397704},
issn = {1098-5336},
abstract = {CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable, genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making targeted, marker-less gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) pathway is being exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid that is temperature-sensitive for replication, by growing it at the non-permissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The here developed approaches could be potentially transported to other Gram-positive bacteria.ImportanceStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing over a million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts in identifying new vaccine candidates. Here, we introduced an easy to use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The here used approaches will advance genome editing projects in this important human pathogen.},
}
@article {pmid33396075,
year = {2021},
author = {Rai, KK and Pandey, N and Meena, RP and Rai, SP},
title = {Biotechnological strategies for enhancing heavy metal tolerance in neglected and underutilized legume crops: A comprehensive review.},
journal = {Ecotoxicology and environmental safety},
volume = {208},
number = {},
pages = {111750},
doi = {10.1016/j.ecoenv.2020.111750},
pmid = {33396075},
issn = {1090-2414},
abstract = {Contamination of agricultural land and water by heavy metals due to rapid industrialization and urbanization including various natural processes have become one of the major constraints to crop growth and productivity. Several studies have reported that to counteract heavy metal stress, plants should be able to maneuver various physiological, biochemical and molecular processes to improve their growth and development under heavy metal stress. With the advent of modern biotechnological tools and techniques it is now possible to tailor legume and other plants overexpressing stress-induced genes, transcription factors, proteins, and metabolites that are directly involved in heavy metal stress tolerance. This review provides an in-depth overview of various biotechnological approaches and/or strategies that can be used for enhancing detoxification of the heavy metals by stimulating phytoremediation processes. Synthetic biology tools involved in the engineering of legume and other crop plants against heavy metal stress tolerance are also discussed herewith some pioneering examples where synthetic biology tools that have been used to modify plants for specific traits. Also, CRISPR based genetic engineering of plants, including their role in modulating the expression of several genes/ transcription factors in the improvement of abiotic stress tolerance and phytoremediation ability using knockdown and knockout strategies has also been critically discussed.},
}
@article {pmid32615024,
year = {2020},
author = {Otte, K and Kühne, NM and Furrer, AD and Baena Lozada, LP and Lutz, VT and Schilling, T and Hertel, R},
title = {A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages.},
journal = {Letters in applied microbiology},
volume = {71},
number = {6},
pages = {588-595},
doi = {10.1111/lam.13349},
pmid = {32615024},
issn = {1472-765X},
support = {94045//Volkswagen Foundation/ ; },
mesh = {Bacillus Phages/*genetics/physiology ; Bacillus subtilis/*virology ; *CRISPR-Cas Systems ; Genetic Engineering ; Mutagenesis ; RNA, Guide/genetics/metabolism ; },
abstract = {Here, we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.},
}
@article {pmid32488146,
year = {2020},
author = {Schwinn, MK and Steffen, LS and Zimmerman, K and Wood, KV and Machleidt, T},
title = {A Simple and Scalable Strategy for Analysis of Endogenous Protein Dynamics.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {8953},
pmid = {32488146},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Luminescent Measurements/*methods ; Luminescent Proteins/*analysis ; Plasmids ; Proteins/*analysis ; },
abstract = {The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.},
}
@article {pmid32376684,
year = {2020},
author = {Alam, SMD and Tsukamoto, Y and Ogawa, M and Senoo, Y and Ikeda, K and Tashima, Y and Takeuchi, H and Okajima, T},
title = {N-Glycans on EGF domain-specific O-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {25},
pages = {8560-8574},
pmid = {32376684},
issn = {1083-351X},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Chromatography, High Pressure Liquid ; Endoplasmic Reticulum/*metabolism ; Endoplasmic Reticulum Stress/drug effects ; Gene Editing ; Glycopeptides/analysis ; Glycosylation ; Humans ; Mice ; Mutagenesis, Site-Directed ; N-Acetylglucosaminyltransferases/deficiency/genetics/*metabolism ; Receptor, Notch1/genetics/metabolism ; Sequence Alignment ; Tandem Mass Spectrometry ; Tunicamycin/pharmacology ; },
abstract = {Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.},
}
@article {pmid32371392,
year = {2020},
author = {Chen, H and Shi, Z and Guo, J and Chang, KJ and Chen, Q and Yao, CH and Haigis, MC and Shi, Y},
title = {The human mitochondrial 12S rRNA m4C methyltransferase METTL15 is required for mitochondrial function.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {25},
pages = {8505-8513},
pmid = {32371392},
issn = {1083-351X},
mesh = {CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/metabolism ; Evolution, Molecular ; Gene Editing ; Genome, Mitochondrial ; Glycolysis ; Humans ; Kinetics ; Methylation ; Methyltransferases/genetics/*metabolism ; Microscopy, Fluorescence ; Mitochondria/genetics/*metabolism ; RNA, Messenger/metabolism ; RNA, Mitochondrial/metabolism ; RNA, Ribosomal/genetics/*metabolism ; Substrate Specificity ; },
abstract = {Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes.},
}
@article {pmid32358066,
year = {2020},
author = {Melville, DB and Studer, S and Schekman, R},
title = {Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {25},
pages = {8401-8412},
pmid = {32358066},
issn = {1083-351X},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Binding Sites ; CRISPR-Cas Systems/genetics ; Cell Line ; Dimerization ; Gene Editing ; Guanosine Triphosphate/chemistry/metabolism ; Humans ; Molecular Dynamics Simulation ; Monomeric GTP-Binding Proteins/chemistry/deficiency/genetics/*metabolism ; Phylogeny ; Protein Binding ; Protein Conformation, alpha-Helical ; Recombinant Fusion Proteins/biosynthesis/chemistry/isolation &amp; purification ; Sequence Alignment ; Vesicular Transport Proteins/chemistry/*metabolism ; },
abstract = {Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog-specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.},
}
@article {pmid32299216,
year = {2020},
author = {Huang, L and Tian, H and Luo, J and Song, N and Wu, J},
title = {CRISPR/Cas9 Based Knockout of miR-145 Affects Intracellular Fatty Acid Metabolism by Targeting INSIG1 in Goat Mammary Epithelial Cells.},
journal = {Journal of agricultural and food chemistry},
volume = {68},
number = {18},
pages = {5138-5146},
doi = {10.1021/acs.jafc.0c00845},
pmid = {32299216},
issn = {1520-5118},
mesh = {Animals ; CRISPR-Cas Systems ; Epithelial Cells/*metabolism ; Fatty Acids/*metabolism ; Female ; Gene Knockout Techniques ; Goats/*genetics/metabolism ; Intracellular Signaling Peptides and Proteins/*genetics/metabolism ; Mammary Glands, Animal/*metabolism ; MicroRNAs/*genetics/metabolism ; },
abstract = {MiR-145 modulates fatty acid metabolism by regulating the expression of fatty acid metabolism-related genes in goat mammary epithelial cells. Previous studies using RNAi methods have clarified the function of miR-145 in lipogenesis. However, there are limiting factors such as short-term and inconsistent inhibition efficiency in RNAi method. On the basis of previous miR-145 functional studies, this study aims to knock out miR-145 and validate the function using CRISPR/Cas9 technology. We successfully obtained the single cell clone which had single nucleotide deletion around the Drosha processing site. The expression of miR-145 was significantly decreased, and the mRNA and protein expression of target gene INSIG1 were both increased by RT-qPCR and Western blot. The expression of fatty acid metabolism-associated gene (DGAT1, AGPAT6, TIP47, ADFP, CD36, ACSL1, ATGL, ACOX, CPT1A, FADS2, ELOVL5, PPARA, SCD1, FASN, and ACACA) were decreased. The contents of triacylglycerol and cholesterol were significantly inhibited. The percentage of C17:0 and C18:0 saturated fatty acid increased. Taken together, these data suggested that knockout of miR-145 could inhibit TAG and cholesterol contents and affect fatty acid composition through regulating the expression of fatty acid metabolism-related genes. These findings provide a sufficient theoretical basis for improving goat milk quality by miR-145.},
}
@article {pmid32230903,
year = {2020},
author = {Peddle, CF and Fry, LE and McClements, ME and MacLaren, RE},
title = {CRISPR Interference-Potential Application in Retinal Disease.},
journal = {International journal of molecular sciences},
volume = {21},
number = {7},
pages = {},
pmid = {32230903},
issn = {1422-0067},
support = {NA//Mabel Churn Scholarship/ ; NA//NIHR Oxford Biomedical Research Centre/ ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression ; Gene Knockdown Techniques ; Gene Silencing ; Genetic Therapy ; Humans ; Induced Pluripotent Stem Cells ; RNA Interference ; RNA, Guide/metabolism ; Retinal Diseases/*genetics/*metabolism/therapy ; Transcription, Genetic ; },
abstract = {The treatment of dominantly inherited retinal diseases requires silencing of the pathogenic allele. RNA interference to suppress gene expression suffers from wide-spread off-target effects, while CRISPR-mediated gene disruption creates permanent changes in the genome. CRISPR interference uses a catalytically inactive 'dead' Cas9 directed by a guide RNA to block transcription of chosen genes without disrupting the DNA. It is highly specific and potentially reversible, increasing its safety profile as a therapy. Pre-clinical studies have demonstrated the versatility of CRISPR interference for gene silencing both in vivo and in ex vivo modification of iPSCs for transplantation. Applying CRISPR interference techniques for the treatment of autosomal dominant inherited retinal diseases is promising but there are few in vivo studies to date. This review details how CRISPR interference might be used to treat retinal diseases and addresses potential challenges for clinical translation.},
}
@article {pmid31672582,
year = {2020},
author = {Yang, TC and Chang, CY and Yarmishyn, AA and Mao, YS and Yang, YP and Wang, ML and Hsu, CC and Yang, HY and Hwang, DK and Chen, SJ and Tsai, ML and Lai, YH and Tzeng, Y and Chang, CC and Chiou, SH},
title = {Carboxylated nanodiamond-mediated CRISPR-Cas9 delivery of human retinoschisis mutation into human iPSCs and mouse retina.},
journal = {Acta biomaterialia},
volume = {101},
number = {},
pages = {484-494},
doi = {10.1016/j.actbio.2019.10.037},
pmid = {31672582},
issn = {1878-7568},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Eye Proteins/genetics ; Gene Editing ; *Gene Transfer Techniques ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Male ; Mice, Inbred C57BL ; Mutation/*genetics ; Nanodiamonds/*chemistry ; Photoreceptor Cells, Vertebrate/pathology ; Retina/*metabolism ; Retinoschisis/*genetics ; },
abstract = {Nanodiamonds (NDs) are considered to be relatively safe carbon nanomaterials used for the transmission of DNA, proteins and drugs. The feasibility of utilizing the NDs to deliver CRISPR-Cas9 system for gene editing has not been clearly studied. Therefore, in this study, we aimed to use NDs as the carriers of CRISPR-Cas9 components designed to introduce the mutation in RS1 gene associated with X-linked retinoschisis (XLRS). ND particles with a diameter of 3 nm were functionalized by carboxylation of the surface and covalently conjugated with fluorescent mCherry protein. Two linear DNA constructs were attached to the conjugated mCherry: one encoded Cas9 endonuclease and GFP reporter, another encoded sgRNA and contained insert of HDR template designed to introduce RS1 c.625C>T mutation. Such nanoparticles were successfully delivered and internalized by human iPSCs and mouse retinas, the efficiency of internalization was significantly improved by mixing with BSA. The delivery of ND particles led to introduction of RS1 c.625C>T mutation in both human iPSCs and mouse retinas. Rs1 gene editing in mouse retinas resulted in several pathological features typical for XLRS, such as aberrant photoreceptor structure. To conclude, our ND-based CRISPR-Cas9 delivery system can be utilized as a tool for creating in vitro and in vivo disease models of XLRS. STATEMENT OF SIGNIFICANCE: X-linked retinoschisis (XLRS) is a prevalent hereditary retinal disease, which is caused by mutations in RS1 gene, whose product is important for structural organization of the retina. The recent development of genome editing techniques such as CRISPR-Cas9 significantly improved the prospects for better understanding the pathology and development of treatment for this disease. Firstly, gene editing can allow development of appropriate in vitro and in vivo disease models; secondly, CRISPR-Cas9 can be applied for gene therapy by removing the disease-causative mutation in vivo. The major prerequisite for these approaches is to develop safe and efficient CRISPR-Cas9 delivery system. In this study, we tested specifically modified nanodiamonds for such a delivery system. We were able to introduce Rs1 mutation into the mouse retina and, importantly, observed several XLRS-specific effects.},
}
@article {pmid33346711,
year = {2020},
author = {Xu, X and Luo, T and Gao, J and Lin, N and Li, W and Xia, X and Wang, J},
title = {CRISPR-Assisted DNA Detection: A Novel dCas9-Based DNA Detection Technique.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {487-502},
doi = {10.1089/crispr.2020.0041},
pmid = {33346711},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; DNA, Viral/genetics ; Genetic Engineering/methods ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/*methods ; Nucleic Acid Hybridization/*methods ; Papillomavirus Infections/genetics ; RNA, Guide/genetics/metabolism ; },
abstract = {Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here we developed CRISPR-Cas9-assisted DNA detection (CADD). In this detection, a DNA sample is incubated with a pair of capture single guide RNAs (sgRNAs; sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature (RT) for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa, and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally, the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cells, and viruses. In particular, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples was successfully detected by the method. All detections can be finished in 30 min at RT. This detection holds promise for rapid on-the-spot detection or point-of-care testing.},
}
@article {pmid33311465,
year = {2020},
author = {Das, A and Hand, TH and Smith, CL and Wickline, E and Zawrotny, M and Li, H},
title = {The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6346},
pmid = {33311465},
issn = {2041-1723},
support = {R01 GM099604/GM/NIGMS NIH HHS/United States ; P30 GM124165/GM/NIGMS NIH HHS/United States ; S10 RR029205/RR/NCRR NIH HHS/United States ; S10 OD021527/OD/NIH HHS/United States ; P41 GM111244/GM/NIGMS NIH HHS/United States ; },
mesh = {Actinobacteria/*enzymology/genetics ; Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Protein 9/*chemistry/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crystallography, X-Ray ; Cytosine ; DNA/chemistry/genetics/metabolism ; Gene Editing/*methods ; Genome ; High-Throughput Nucleotide Sequencing ; Methylation ; Models, Molecular ; Protein Conformation ; RNA, Guide/chemistry ; },
abstract = {Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.},
}
@article {pmid33253175,
year = {2020},
author = {Li, R and Xia, X and Wang, X and Sun, X and Dai, Z and Huo, D and Zheng, H and Xiong, H and He, A and Wu, X},
title = {Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model.},
journal = {PLoS biology},
volume = {18},
number = {11},
pages = {e3000749},
pmid = {33253175},
issn = {1545-7885},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Doxycycline/metabolism ; Embryonic Stem Cells/metabolism/physiology ; Gene Editing/*methods ; Gene Expression/genetics ; Gene Silencing/physiology ; Kruppel-Like Transcription Factors/*genetics/metabolism ; Loss of Function Mutation/genetics ; Mice ; Mice, Knockout ; Models, Biological ; RNA, Guide/genetics ; Reproducibility of Results ; Transgenes/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.},
}
@article {pmid33122441,
year = {2020},
author = {Holmes, DL and Vogt, DT and Lagunoff, M},
title = {A CRISPR-Cas9 screen identifies mitochondrial translation as an essential process in latent KSHV infection of human endothelial cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {45},
pages = {28384-28392},
pmid = {33122441},
issn = {1091-6490},
support = {R01 CA189986/CA/NCI NIH HHS/United States ; R01 CA217788/CA/NCI NIH HHS/United States ; R21 CA240479/CA/NCI NIH HHS/United States ; T32 AI083203/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endothelial Cells/metabolism ; Herpesviridae Infections/*genetics ; Herpesvirus 8, Human/*genetics/physiology ; Humans ; Lymphoma, Primary Effusion/genetics ; Mitochondria/*metabolism ; Sarcoma, Kaposi ; Virus Latency/*genetics ; Virus Replication ; },
abstract = {Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The main proliferating component of KS tumors is a cell of endothelial origin termed the spindle cell. Spindle cells are predominantly latently infected with only a small percentage of cells undergoing viral replication. As there is no direct treatment for latent KSHV, identification of host vulnerabilities in latently infected endothelial cells could be exploited to inhibit KSHV-associated tumor cells. Using a pooled CRISPR-Cas9 lentivirus library, we identified host factors that are essential for the survival or proliferation of latently infected endothelial cells in culture, but not their uninfected counterparts. Among the many host genes identified, there was an enrichment in genes localizing to the mitochondria, including genes involved in mitochondrial translation. Antibiotics that inhibit bacterial and mitochondrial translation specifically inhibited the expansion of latently infected endothelial cells and led to increased cell death in patient-derived PEL cell lines. Direct inhibition of mitochondrial respiration or ablation of mitochondrial genomes leads to increased death in latently infected cells. KSHV latent infection decreases mitochondrial numbers, but there are increases in mitochondrial size, genome copy number, and transcript levels. We found that multiple gene products of the latent locus localize to the mitochondria. During latent infection, KSHV significantly alters mitochondrial biology, leading to enhanced sensitivity to inhibition of mitochondrial respiration, which provides a potential therapeutic avenue for KSHV-associated cancers.},
}
@article {pmid33106426,
year = {2020},
author = {Alward, BA and Laud, VA and Skalnik, CJ and York, RA and Juntti, SA and Fernald, RD},
title = {Modular genetic control of social status in a cichlid fish.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {45},
pages = {28167-28174},
pmid = {33106426},
issn = {1091-6490},
support = {R37 NS034950/NS/NINDS NIH HHS/United States ; R01 NS034950/NS/NINDS NIH HHS/United States ; R21 MH096220/MH/NIMH NIH HHS/United States ; R56 NS034950/NS/NINDS NIH HHS/United States ; R03 MH101373/MH/NIMH NIH HHS/United States ; },
mesh = {Androgens/metabolism ; Animals ; CRISPR-Cas Systems ; *Cichlids/genetics/physiology ; Female ; Gene Editing ; *Gene Expression Regulation/genetics/physiology ; Male ; Mutation ; Receptors, Androgen/genetics/physiology ; Social Behavior ; *Social Dominance ; },
abstract = {Social hierarchies are ubiquitous in social species and profoundly influence physiology and behavior. Androgens like testosterone have been strongly linked to social status, yet the molecular mechanisms regulating social status are not known. The African cichlid fish Astatotilapia burtoni is a powerful model species for elucidating the role of androgens in social status given their rich social hierarchy and genetic tractability. Dominant A. burtoni males possess large testes and bright coloration and perform aggressive and reproductive behaviors while nondominant males do not. Social status in A. burtoni is in flux, however, as males alter their status depending on the social environment. Due to a teleost-specific whole-genome duplication, A. burtoni possess two androgen receptor (AR) paralogs, ARα and ARβ, providing a unique opportunity to disentangle the role of gene duplication in the evolution of social systems. Here, we used CRISPR/Cas9 gene editing to generate AR mutant A. burtoni and performed a suite of experiments to interrogate the mechanistic basis of social dominance. We find that ARβ, but not ARα, is required for testes growth and bright coloration, while ARα, but not ARβ, is required for the performance of reproductive behavior and aggressive displays. Both receptors are required to reduce flees from females and either AR is sufficient for attacking males. Thus, social status in A. burtoni is inordinately dissociable and under the modular control of two AR paralogs. This type of nonredundancy may be important in facilitating social plasticity in A. burtoni and other species whose social status relies on social experience.},
}
@article {pmid33097661,
year = {2020},
author = {Wei, X and Yang, J and Adair, SJ and Ozturk, H and Kuscu, C and Lee, KY and Kane, WJ and O'Hara, PE and Liu, D and Demirlenk, YM and Habieb, AH and Yilmaz, E and Dutta, A and Bauer, TW and Adli, M},
title = {Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {45},
pages = {28068-28079},
pmid = {33097661},
issn = {1091-6490},
support = {P30 CA044579/CA/NCI NIH HHS/United States ; R01 CA060499/CA/NCI NIH HHS/United States ; R01 CA211648/CA/NCI NIH HHS/United States ; R25 CA206972/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/drug effects ; Deoxycytidine/*analogs &amp; derivatives/pharmacology ; Drug Development ; Gene Knockout Techniques ; Humans ; Mice, Nude ; Pancreatic Neoplasms/*metabolism ; *Protein-Arginine N-Methyltransferases/antagonists &amp; inhibitors/genetics/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.},
}
@article {pmid32967957,
year = {2020},
author = {Huang, Y and Lin, Q and Huo, Z and Chen, C and Zhou, S and Ma, X and Gao, H and Lin, Y and Li, X and He, J and Zhang, P and Liu, C},
title = {Inositol-Requiring Enzyme 1α Promotes Zika Virus Infection through Regulation of Stearoyl Coenzyme A Desaturase 1-Mediated Lipid Metabolism.},
journal = {Journal of virology},
volume = {94},
number = {23},
pages = {},
pmid = {32967957},
issn = {1098-5514},
mesh = {A549 Cells ; Animals ; Brain/pathology/virology ; CRISPR-Cas Systems ; Cell Line ; Disease Models, Animal ; Endoplasmic Reticulum/metabolism ; Endoribonucleases/genetics/*metabolism ; Gene Editing ; Gene Knockout Techniques ; Humans ; Inositol/*metabolism ; Lipid Metabolism/*physiology ; Mice ; Oleic Acid/metabolism ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; Stearoyl-CoA Desaturase/genetics/*metabolism ; Unfolded Protein Response ; Virus Replication/physiology ; Zika Virus/*metabolism ; Zika Virus Infection/*metabolism/pathology ; },
abstract = {Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which has become a global epidemic threat due to its rapid spread and association with serious consequences of infection, including neonatal microcephaly. Inositol-requiring enzyme 1α (IRE1α) is an endoplasmic reticulum (ER)-related transmembrane protein that mediates unfolded protein response (UPR) pathway and has been indicated to play an important role in flavivirus replication. However, the mechanism of how IRE1α affects ZIKV replication remains unknown. In this study, we explored the role of IRE1α in ZIKV infection in vitro and in vivo by using CRISPR/Cas9-based gene knockout and RNA interference-based gene knockdown techniques. Both knockout and knockdown of IRE1α dramatically reduced ZIKV replication levels, including viral RNA levels, protein expression, and titers in different human cell lines. Trans-complementation with IRE1α restored viral replication levels decreased by IRE1α depletion. Furthermore, the proviral effect of IRE1α was dependent on its kinase and RNase activities. Importantly, we found that IRE1α promoted the replication of ZIKV through upregulating the accumulation of monounsaturated fatty acid (MUFA) rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1), which further affected the production of oleic acid (OA) and lipid droplet. Finally, our data demonstrated that in the brain tissues of ZIKV-infected mice, the replication levels of ZIKV and virus-related lesions were significantly suppressed by both the kinase and RNase inhibitors of IRE1α. Taken together, our results identified IRE1α as a ZIKV dependency factor which promotes viral replication through affecting SCD1-mediated lipid metabolism, potentially providing a novel molecular target for the development of anti-ZIKV agents.IMPORTANCE Zika virus (ZIKV) has been linked to serious neurologic disorders and causes widespread concern in the field of global public health. Inositol requiring enzyme 1α (IRE1α) is an ER-related transmembrane protein that mediates unfolded protein response (UPR) pathway. Here, we revealed that IRE1α is a proviral factor for ZIKV replication both in culture cells and mice model, which relies on its kinase and RNase activities. Importantly, we further provided evidence that upon ZIKV infection, IRE1α is activated and splices XBP1 mRNA which enhances the expression of monounsaturated fatty acids rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1) and subsequent lipid droplet production. Our data uncover a novel mechanism of IRE1α proviral effect by modulating lipid metabolism, providing the first evidence of a close relationship between IRE1α-mediated UPR, lipid metabolism, and ZIKV replication and indicating IRE1α inhibitors as potentially effective anti-ZIKV agents.},
}
@article {pmid32907973,
year = {2020},
author = {Kawagishi, T and Kanai, Y and Nouda, R and Fukui, I and Nurdin, JA and Matsuura, Y and Kobayashi, T},
title = {Generation of Genetically RGD σ1-Modified Oncolytic Reovirus That Enhances JAM-A-Independent Infection of Tumor Cells.},
journal = {Journal of virology},
volume = {94},
number = {23},
pages = {},
pmid = {32907973},
issn = {1098-5514},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Cell Adhesion Molecules ; Cell Line, Tumor ; Gene Knockout Techniques ; Humans ; Junctional Adhesion Molecule A/*genetics/*metabolism ; Mammalian orthoreovirus 3/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Nude ; Oligopeptides/*metabolism ; Oncolytic Virotherapy ; Oncolytic Viruses/genetics ; Orthoreovirus/genetics/metabolism ; Receptors, Cell Surface ; Reoviridae/*genetics/*metabolism ; Virus Replication ; },
abstract = {Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine-glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy.IMPORTANCE Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus Orthoreovirus, family Reoviridae, is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1.},
}
@article {pmid32843651,
year = {2020},
author = {Zou, ZV and Gul, N and Lindberg, M and Bokhari, AA and Eklund, EM and Garellick, V and Patel, AAH and Dzanan, JJ and Titmuss, BO and Le Gal, K and Johansson, I and Tivesten, Å and Forssell-Aronsson, E and Bergö, MO and Staffas, A and Larsson, E and Sayin, VI and Lindahl, P},
title = {Genomic profiling of the transcription factor Zfp148 and its impact on the p53 pathway.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14156},
pmid = {32843651},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Checkpoints/genetics ; Cell Cycle Proteins/biosynthesis/genetics ; Cell Division ; Cell Line ; Chromatin Immunoprecipitation ; Cisplatin/toxicity ; Cyclin-Dependent Kinase Inhibitor p16/metabolism ; DNA Damage ; DNA-Binding Proteins/deficiency/*genetics/physiology ; Down-Regulation ; E2F Transcription Factors/physiology ; Etoposide/toxicity ; Fibroblasts ; Gene Expression Regulation/*genetics ; Gene Ontology ; Mice ; RNA Interference ; RNA, Small Interfering/genetics ; Signal Transduction/*genetics ; Transcription Factors/deficiency/*genetics/physiology ; Tumor Suppressor Protein p53/*physiology ; },
abstract = {Recent data suggest that the transcription factor Zfp148 represses activation of the tumor suppressor p53 in mice and that therapeutic targeting of the human orthologue ZNF148 could activate the p53 pathway without causing detrimental side effects. We have previously shown that Zfp148 deficiency promotes p53-dependent proliferation arrest of mouse embryonic fibroblasts (MEFs), but the underlying mechanism is not clear. Here, we showed that Zfp148 deficiency downregulated cell cycle genes in MEFs in a p53-dependent manner. Proliferation arrest of Zfp148-deficient cells required increased expression of ARF, a potent activator of the p53 pathway. Chromatin immunoprecipitation showed that Zfp148 bound to the ARF promoter, suggesting that Zfp148 represses ARF transcription. However, Zfp148 preferentially bound to promoters of other transcription factors, indicating that deletion of Zfp148 may have pleiotropic effects that activate ARF and p53 indirectly. In line with this, we found no evidence of genetic interaction between TP53 and ZNF148 in CRISPR and siRNA screen data from hundreds of human cancer cell lines. We conclude that Zfp148 deficiency, by increasing ARF transcription, downregulates cell cycle genes and cell proliferation in a p53-dependent manner. However, the lack of genetic interaction between ZNF148 and TP53 in human cancer cells suggests that therapeutic targeting of ZNF148 may not increase p53 activity in humans.},
}
@article {pmid32810562,
year = {2020},
author = {Yokoyama, M and Matsuzawa, T and Yoshikawa, T and Nunomiya, A and Yamaguchi, Y and Yanai, K},
title = {Heparan sulfate controls skeletal muscle differentiation and motor functions.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1864},
number = {12},
pages = {129707},
doi = {10.1016/j.bbagen.2020.129707},
pmid = {32810562},
issn = {1872-8006},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Heparitin Sulfate/antagonists &amp; inhibitors/genetics/*metabolism ; Mice ; Motor Activity ; *Muscle Development ; Muscle, Skeletal/cytology/*physiology ; Myoblasts/*cytology/metabolism ; },
abstract = {BACKGROUND: Heparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear.<br><br>METHODS: We examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system.<br><br>RESULTS: CRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl2 injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells.<br><br>SIGNIFICANCE: These results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.},
}
@article {pmid32719542,
year = {2020},
author = {Cai, EP and Ishikawa, Y and Zhang, W and Leite, NC and Li, J and Hou, S and Kiaf, B and Hollister-Lock, J and Yilmaz, NK and Schiffer, CA and Melton, DA and Kissler, S and Yi, P},
title = {Genome-scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes.},
journal = {Nature metabolism},
volume = {2},
number = {9},
pages = {934-945},
doi = {10.1038/s42255-020-0254-1},
pmid = {32719542},
issn = {2522-5812},
support = {T32 DK007260/DK/NIDDK NIH HHS/United States ; P30 DK036836/DK/NIDDK NIH HHS/United States ; R01 DK120445/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Autoimmunity/drug effects ; *CRISPR-Cas Systems ; Diabetes Mellitus, Type 1/*drug therapy/immunology/pathology ; Endoplasmic Reticulum Stress ; Enzyme Inhibitors/pharmacology ; Female ; *Genome-Wide Association Study ; Induced Pluripotent Stem Cells/immunology ; Insulin-Secreting Cells/*drug effects/immunology/pathology ; Islets of Langerhans Transplantation ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Mice, Knockout ; Monoamine Oxidase/*drug effects ; Mutation ; Pargyline/pharmacology ; },
abstract = {Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Pluripotent stem cells can now be differentiated into beta cells, thus raising the prospect of a cell replacement therapy for T1D. However, autoimmunity would rapidly destroy newly transplanted beta cells. Using a genome-scale CRISPR screen in a mouse model for T1D, we show that deleting RNLS, a genome-wide association study candidate gene for T1D, made beta cells resistant to autoimmune killing. Structure-based modelling identified the U.S. Food and Drug Administration-approved drug pargyline as a potential RNLS inhibitor. Oral pargyline treatment protected transplanted beta cells in diabetic mice, thus leading to disease reversal. Furthermore, pargyline prevented or delayed diabetes onset in several mouse models for T1D. Our results identify RNLS as a modifier of beta cell vulnerability and as a potential therapeutic target to avert beta cell loss in T1D.},
}
@article {pmid32621079,
year = {2020},
author = {Chang, AX and Chen, B and Yang, AG and Hu, RS and Feng, QF and Chen, M and Yang, XN and Luo, CG and Li, YY and Wang, YY},
title = {The trichome-specific acetolactate synthase NtALS1 gene, is involved in acylsugar biosynthesis in tobacco (Nicotiana tabacum L.).},
journal = {Planta},
volume = {252},
number = {1},
pages = {13},
doi = {10.1007/s00425-020-03418-x},
pmid = {32621079},
issn = {1432-2048},
support = {1610232017006//Fundamental Research Funds for Central Non-profit Scientific Institution/ ; 1610232020001//Fundamental Research Funds for Central Non-profit Scientific Institution/ ; },
mesh = {Acetolactate Synthase/*genetics/metabolism ; Arabidopsis Proteins/genetics ; CRISPR-Cas Systems ; Chloroplasts/enzymology ; Diploidy ; Gene Expression Regulation, Plant ; Phylogeny ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Sugars/*metabolism ; Tobacco/genetics/*metabolism ; Trichomes/*enzymology/genetics ; },
abstract = {MAIN CONCLUSION: NtALS1 is specifically expressed in glandular trichomes, and can improve the content of acylsugars in tobacco.<br><br>ABTRACT: The glandular trichomes of many species in the Solanaceae family play an important role in plant defense. These epidermal outgrowths exhibit specialized secondary metabolism, including the production of structurally diverse acylsugars that function in defense against insects and have substantial developmental potential for commercial uses. However, our current understanding of genes involved in acyl chain biosynthesis of acylsugars remains poor in tobacco. In this study, we identified three acetolactate synthase (ALS) genes in tobacco through homology-based gene prediction using Arabidopsis ALS. Quantitative real-time PCR (qRT-PCR) and tissue distribution analyses suggested that NtALS1 was highly expressed in the tips of glandular trichomes. Subcellular localization analysis showed that the NtALS1 localized to the chloroplast. Moreover, in the wild-type K326 variety background, we generated two ntals1 loss-of-function mutants using the CRISPR-Cas9 system. Acylsugars contents in the two ntals1 mutants were significantly lower than those in the wild type. Through phylogenetic tree analysis, we also identified NtALS1 orthologs that may be involved in acylsugar biosynthesis in other Solanaceae species. Taken together, these findings indicate a functional role for NtALS1 in acylsugar biosynthesis in tobacco.},
}
@article {pmid32450806,
year = {2020},
author = {Tian, Y and Chen, K and Li, X and Zheng, Y and Chen, F},
title = {Design of high-oleic tobacco (Nicotiana tabacum L.) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2-2.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {233},
pmid = {32450806},
issn = {1471-2229},
support = {2016ZX08010001-010//Ministry of Agriculture/ ; },
mesh = {*CRISPR-Cas Systems ; Fatty Acid Desaturases/*genetics/metabolism ; Gene Knockout Techniques ; Oleic Acids/chemistry ; Plant Oils/analysis/*chemistry ; Plant Proteins/*genetics/metabolism ; Seeds/chemistry/metabolism ; Tobacco/chemistry/enzymology/*genetics ; },
abstract = {BACKGROUND: Tobacco seed oil could be used as an appropriate feedstock for biodiesel production. However, the high linoleic acid content of tobacco seed oil makes it susceptible to oxidation. Altering the fatty acid profile by increasing the content of oleic acid could improve the properties of biodiesel produced from tobacco seed oil.<br><br>RESULTS: Four FAD2 genes, NtFAD2-1a, NtFAD2-1b, NtFAD2-2a, and NtFAD2-2b, were identified in allotetraploid tobacco genome. Phylogenetic analysis of protein sequences showed that NtFAD2-1a and NtFAD2-2a originated from N. tomentosiformis, while NtFAD2-1b and NtFAD2-2b from N. sylvestris. Expression analysis revealed that NtFAD2-2a and NtFAD2-2b transcripts were more abundant in developing seeds than in other tissues, while NtFAD2-1a and NtFAD2-1b showed low transcript levels in developing seed. Phylogenic analysis showed that NtFAD2-2a and NtFAD2-2b were seed-type FAD2 genes. Heterologous expression in yeast cells demonstrated that both NtFAD2-2a and NtFAD2-2b protein could introduce a double bond at the Δ12 position of fatty acid chain. The fatty acid profile analysis of tobacco fad2-2 mutant seeds derived from CRISPR-Cas9 edited plants showed dramatic increase of oleic acid content from 11% to over 79%, whereas linoleic acid decreased from 72 to 7%. In addition, the fatty acid composition of leaf was not affected in fad2-2 mutant plants.<br><br>CONCLUSION: Our data showed that knockout of seed-type FAD2 genes in tobacco could significantly increase the oleic acid content in seed oil. This research suggests that CRISPR-Cas9 system offers a rapid and highly efficient method in the tobacco seed lipid engineering programs.},
}
@article {pmid32423474,
year = {2020},
author = {Zhang, Y and Held, MA and Showalter, AM},
title = {Elucidating the roles of three β-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {221},
pmid = {32423474},
issn = {1471-2229},
mesh = {Amino Acid Sequence ; Arabidopsis/enzymology/*genetics ; *CRISPR-Cas Systems ; Galactans/*metabolism ; },
abstract = {BACKGROUND: Arabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants. AGPs are implicated in diverse biological processes such as plant growth, development, reproduction, and stress responses. AGPs are extensively glycosylated by the addition of type II arabinogalactan (AG) polysaccharides to hydroxyproline residues in their protein cores. Glucuronic acid (GlcA) is the only negatively charged sugar added to AGPs and the functions of GlcA residues on AGPs remain to be elucidated.<br><br>RESULTS: Three members of the CAZy GT14 family (GLCAT14A-At5g39990, GLCAT14B-At5g15050, and GLCAT14C-At2g37585), which are responsible for transferring glucuronic acid (GlcA) to AGPs, were functionally characterized using a CRISPR/Cas9 gene editing approach in Arabidopsis. RNA seq and qRT-PCR data showed all three GLCAT genes were broadly expressed in different plant tissues, with GLCAT14A and GLCAT14B showing particularly high expression in the micropylar endosperm. Biochemical analysis of the AGPs from knock-out mutants of various glcat single, double, and triple mutants revealed that double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, AGPs isolated from all the glcat mutants displayed significant reductions in calcium binding compared to WT. Further phenotypic analyses found that the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants exhibited significant delays in seed germination, reductions in root hair length, reductions in trichome branching, and accumulation of defective pollen grains. Additionally, both glcat14b glcat14c and glcat14a glcat14b glcat14c displayed significantly shorter siliques and reduced seed set. Finally, all higher-order mutants exhibited significant reductions in adherent seed coat mucilage.<br><br>CONCLUSIONS: This research provides genetic evidence that GLCAT14A-C function in the transfer of GlcA to AGPs, which in turn play a role in a variety of biochemical and physiological phenotypes including calcium binding by AGPs, seed germination, root hair growth, trichome branching, pollen development, silique development, seed set, and adherent seed coat mucilage accumulation.},
}
@article {pmid32413320,
year = {2020},
author = {Bowling, S and Sritharan, D and Osorio, FG and Nguyen, M and Cheung, P and Rodriguez-Fraticelli, A and Patel, S and Yuan, WC and Fujiwara, Y and Li, BE and Orkin, SH and Hormoz, S and Camargo, FD},
title = {An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells.},
journal = {Cell},
volume = {181},
number = {6},
pages = {1410-1422.e27},
doi = {10.1016/j.cell.2020.04.048},
pmid = {32413320},
issn = {1097-4172},
support = {F31 CA235893/CA/NCI NIH HHS/United States ; R01 HL128850/HL/NHLBI NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; P01 HL131477/HL/NHLBI NIH HHS/United States ; U54 DK110805/DK/NIDDK NIH HHS/United States ; R01 DK123216/DK/NIDDK NIH HHS/United States ; R00 GM118910/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Lineage/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Female ; Flow Cytometry/methods ; Hematopoietic Stem Cells/physiology ; Male ; Mice ; Transcriptome/*genetics ; Transduction, Genetic/methods ; },
abstract = {Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.},
}
@article {pmid32302591,
year = {2020},
author = {Roth, TL and Li, PJ and Blaeschke, F and Nies, JF and Apathy, R and Mowery, C and Yu, R and Nguyen, MLT and Lee, Y and Truong, A and Hiatt, J and Wu, D and Nguyen, DN and Goodman, D and Bluestone, JA and Ye, CJ and Roybal, K and Shifrut, E and Marson, A},
title = {Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies.},
journal = {Cell},
volume = {181},
number = {3},
pages = {728-744.e21},
doi = {10.1016/j.cell.2020.03.039},
pmid = {32302591},
issn = {1097-4172},
support = {F30 DK120213/DK/NIDDK NIH HHS/United States ; T32 DK007418/DK/NIDDK NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; L30 AI113413/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; S10 OD021822/OD/NIH HHS/United States ; R01 DK119979/DK/NIDDK NIH HHS/United States ; P50 AI150476/AI/NIAID NIH HHS/United States ; DP2 DA042423/DA/NIDA NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Blood Cells ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Knock-In Techniques/*methods ; Genetic Engineering/*methods ; Humans ; Immunotherapy/*methods ; Mice ; Mice, Inbred NOD ; Mice, SCID ; RNA, Guide/genetics ; Single-Cell Analysis/methods ; T-Lymphocytes ; Transcriptome/genetics ; },
abstract = {Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor β (TGF-β) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.},
}
@article {pmid32276113,
year = {2020},
author = {Zhu, GH and Chereddy, SCRR and Howell, JL and Palli, SR},
title = {Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation.},
journal = {Insect biochemistry and molecular biology},
volume = {122},
number = {},
pages = {103373},
doi = {10.1016/j.ibmb.2020.103373},
pmid = {32276113},
issn = {1879-0240},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques/*instrumentation ; Larva/genetics/growth &amp; development/metabolism ; RNA, Guide/*genetics ; Spodoptera/*genetics/growth &amp; development/metabolism ; },
abstract = {The CRISPR/Cas9 system is an efficient genome editing method that can be used in functional genomics research. The fall armyworm, Spodoptera frugiperda, is a serious agricultural pest that has spread over most of the world. However, very little information is available on functional genomics for this insect. We performed CRISPR/Cas9-mediated site-specific mutagenesis of three target genes: two marker genes [Biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2) and tryptophan 2, 3-dioxygenase (TO)], and a developmental gene, E93 (a key ecdysone-induced transcription factor that promotes adult development). The knockouts (KO) of BLOS2, TO and E93 induced translucent mosaic integument, olive eye color, and larval-pupal intermediate phenotypes, respectively. Sequencing RNA isolated from wild-type and E93 KO insects showed that E93 promotes adult development by influencing the expression of the genes coding for transcription factor, Krüppel homolog 1, the pupal specifier, Broad-Complex, serine proteases, and heat shock proteins. Often, gene-edited insects display mosaicism in which only a fraction of the cells are edited as intended, and establishing a homozygous line is both costly and time-consuming. To overcome these limitations, a method to completely KO the target gene in S. frugiperda by injecting the Cas9 protein and multiple sgRNAs targeting one exon of the E93 gene into embryos was developed. Ten percent of the G0 larvae exhibited larval-pupal intermediates. The mutations were confirmed by T7E1 assay, and the mutation frequency was determined as >80%. Complete KO of the E93 gene was achieved in one generation using the multiple sgRNA method, demonstrating a powerful approach to improve genome editing in lepidopteran and other non-model insects.},
}
@article {pmid32272060,
year = {2020},
author = {Zhou, H and Su, J and Hu, X and Zhou, C and Li, H and Chen, Z and Xiao, Q and Wang, B and Wu, W and Sun, Y and Zhou, Y and Tang, C and Liu, F and Wang, L and Feng, C and Liu, M and Li, S and Zhang, Y and Xu, H and Yao, H and Shi, L and Yang, H},
title = {Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice.},
journal = {Cell},
volume = {181},
number = {3},
pages = {590-603.e16},
doi = {10.1016/j.cell.2020.03.024},
pmid = {32272060},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems/physiology ; Cell Differentiation/physiology ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disease Models, Animal ; Dopamine/metabolism ; Gene Expression Regulation/genetics ; Heterogeneous-Nuclear Ribonucleoproteins/genetics/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Nervous System Diseases/metabolism ; Neurogenesis/*physiology ; Neuroglia/*metabolism ; Neurons/metabolism ; Parkinson Disease/metabolism ; Polypyrimidine Tract-Binding Protein/genetics/metabolism ; Retinal Ganglion Cells/*metabolism/physiology ; },
abstract = {Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.},
}
@article {pmid32243905,
year = {2020},
author = {Huang 黄镜梅, JM and Rao 饶聪, C and Wang 王帅, S and He 何林凤, LF and Zhao 赵思琪, SQ and Zhou 周丽琪, LQ and Zhao 赵云霞, YX and Yang 杨凤霞, FX and Gao 高聪芬, CF and Wu 吴顺凡, SF},
title = {Multiple target-site mutations occurring in lepidopterans confer resistance to diamide insecticides.},
journal = {Insect biochemistry and molecular biology},
volume = {121},
number = {},
pages = {103367},
doi = {10.1016/j.ibmb.2020.103367},
pmid = {32243905},
issn = {1879-0240},
mesh = {Amino Acid Sequence ; Animals ; Benzamides/pharmacology ; CRISPR-Cas Systems ; Drosophila melanogaster/genetics/metabolism ; Insect Proteins/chemistry/*genetics/metabolism ; Insecticide Resistance/*genetics ; Insecticides/*pharmacology ; Moths/drug effects/*genetics/metabolism ; *Mutation ; Pyrazoles/pharmacology ; Ryanodine Receptor Calcium Release Channel/chemistry/*genetics/metabolism ; Sequence Alignment ; Sulfones/pharmacology ; ortho-Aminobenzoates/pharmacology ; },
abstract = {Diamide resistant phenotypes have evolved in the field and the resistance has been attributed to target-site mutations in some lepidopteran pests. In this study, we documented the resistance status of Chilo suppressalis to chlorantraniliprole during 2016-2018 in seven provinces of China. To investigate the possible role of target-site mutations as known from lepidopterans, we sequenced respective domains of the RyR gene of C. suppressalis with different levels of diamide resistance. The results revealed that I4758M (corresponding to I4790M in P. xylostella), Y4667D/C (numbered according to C. suppressalis), G4915E (corresponding to G4946E in P. xylostella), and one novel Y4891F (numbered according to C. suppressalis) RyR target-site mutations were present. The contribution of these mutations was further investigated by diamide toxicity bioassays with eight genome modified Drosophila melanogaster lines. The study showed that genome modified flies bearing the Y4667D mutation (corresponding to the Y4667D and I4758M simultaneous mutation in C. suppressalis) exhibited high resistance ratios to chlorantraniliprole (1542.8-fold), cyantraniliprole (487.9-fold) and tetrachlorantraniliprole (290.1-fold). The M4758I and G4915E simultaneous mutations (corresponding to single G4915E mutation in C. suppressalis) showed high resistance ratios to chlorantraniliprole (153.1-fold) and cyantraniliprole (323.5-fold), and relatively low resistance to flubendiamide (28.9-fold) and tetrachlorantraniliprole (25.2-fold). These findings suggest that multiple point mutations in RyR confer diamide resistance of C. suppressalis. The results contribute to a better understanding of insect diamide resistance mechanisms and provide insights on the impact of RyR target-site mutations in insects.},
}
@article {pmid32243904,
year = {2020},
author = {Zhang, R and Zhang, Z and Huang, Y and Qian, A and Tan, A},
title = {A single ortholog of teashirt and tiptop regulates larval pigmentation and adult appendage patterning in Bombyx mori.},
journal = {Insect biochemistry and molecular biology},
volume = {121},
number = {},
pages = {103369},
doi = {10.1016/j.ibmb.2020.103369},
pmid = {32243904},
issn = {1879-0240},
mesh = {Animals ; Bombyx/anatomy &amp; histology/*genetics/growth &amp; development/metabolism ; *CRISPR-Cas Systems ; Extremities/anatomy &amp; histology ; *Gain of Function Mutation ; Insect Proteins/*genetics/metabolism ; Larva/genetics/growth &amp; development/physiology ; *Loss of Function Mutation ; Pigmentation/genetics ; },
abstract = {Two paralogous genes, teashirt (tsh) and tiptop (tio), encode zinc-finger transcription factors and play important roles in insect growth and development. In the fruit fly, Drosophila melanogaster, tsh promotes trunk segmental identities and contributes to the patterning of other tissues during the embryonic stage. During the adult stage, tsh contributes to the specification and patterning of appendages, including the leg, wing and eye. While tio acts redundantly with tsh, flies lacking tio function are viable without deleterious phenotypes. This gene pair is present in the genomes of all Drosophila species but only as a single homologue in several other insect species. In Oncopeltus fasciatus and Tribolium castaneum, tsh/tio has been functionally characterized as specifying the identity of the leg during the adult stage. However, in lepidopteran insects which include large numbers of pests in agriculture and forestry, as well as the major silk producer silkworm Bombyx mori, the biological functions of tsh/tio are still poorly understood. In the current study, we performed functional analysis of tsh/tio by using both CRISPR/Cas9-mediated mutagenesis and transposon-mediated ectopic expression in B. mori. The results show that loss of tsh/tio function affected pigmentation during the larval stage and appendage pattering during the adult stage. RNA-seq analysis and subsequent q-RT-PCR analysis revealed that depletion of tsh/tio significantly elevated the expression of the kynurenine 3-monooxygenase gene, as well as melanin synthase-related genes during the larval stage. Furthermore, ubiquitous ectopic expression of tsh/tio induces developmental retardation and eventually larval lethality. These data reveal evolutionarily conserved functions of tsh/tio in controlling adult appendage patterning, as well as the novel function of regulating larval pigmentation in B. mori, providing novel insights into how tsh/tio regulates insect growth and development.},
}
@article {pmid32221665,
year = {2020},
author = {Zhi, J and Liu, X and Li, D and Huang, Y and Yan, S and Cao, B and Qiu, Z},
title = {CRISPR/Cas9-mediated SlAN2 mutants reveal various regulatory models of anthocyanin biosynthesis in tomato plant.},
journal = {Plant cell reports},
volume = {39},
number = {6},
pages = {799-809},
doi = {10.1007/s00299-020-02531-1},
pmid = {32221665},
issn = {1432-203X},
support = {31801863//National Natural Science Foundation of China/ ; 2018A030310211//Natural Science Foundation of Guangdong Province/ ; },
mesh = {Anthocyanins/*biosynthesis/*genetics ; *CRISPR-Cas Systems ; Cotyledon/genetics/metabolism ; Fruit/*genetics/*metabolism ; Gene Expression Regulation, Plant ; Hypocotyl/genetics/metabolism ; Lycopersicon esculentum/*genetics/metabolism ; Mutation ; Phenotype ; Plant Development ; Plant Proteins/*genetics ; Plants, Genetically Modified ; Sequence Alignment ; Sequence Analysis, DNA ; Transcription Factors/*genetics ; Transcriptome ; Transformation, Genetic ; },
abstract = {KEY MESSAGE: Combining phenotype and gene expression analysis of the CRISPR/Cas9-induced SlAN2 mutants, we revealed that SlAN2 specifically regulated anthocyanin accumulation in vegetative tissues in purple tomato cultivar 'Indigo Rose.' Anthocyanins play an important role in plant development and also exhibit human health benefits. The tomato genome contains four highly homologous anthocyanin-related R2R3-MYB transcription factors: SlAN2, SlANT1, SlANT1-like, and SlAN2-like/Aft. SlAN2-like/Aft regulates anthocyanin accumulation in the fruit; however, the genetic function of the other three factors remains unclear. To better understand the function of R2R3-MYB transcription factors, we conducted targeted mutagenesis of SlAN2 in the purple tomato cultivar 'Indigo Rose' using clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The SlAN2 mutants had a fruit color and anthocyanin content similar to cv. 'Indigo Rose,' while the anthocyanin content and the relative expression levels of several anthocyanin-related genes in vegetative tissues were significantly lower in the SlAN2 mutant relative to cv. Indigo Rose. Furthermore, we found that anthocyanin biosynthesis is controlled by different regulators between tomato hypocotyls and cotyledons. In addition, SlAN2 mutants were shorter, with smaller and lighter fruits than cv. 'Indigo Rose.' Our findings further our understanding of anthocyanin production in tomato and other plant species.},
}
@article {pmid32197890,
year = {2020},
author = {Zheng, C and Schneider, JW and Hsieh, J},
title = {Role of RB1 in human embryonic stem cell-derived retinal organoids.},
journal = {Developmental biology},
volume = {462},
number = {2},
pages = {197-207},
pmid = {32197890},
issn = {1095-564X},
support = {R01 NS089770/NS/NINDS NIH HHS/United States ; R01 NS093992/NS/NINDS NIH HHS/United States ; R01 NS113516/NS/NINDS NIH HHS/United States ; R21 AG066496/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Apoptosis/physiology ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Human Embryonic Stem Cells/cytology/*metabolism ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Organoids/cytology ; Pluripotent Stem Cells/cytology ; Retina/*embryology/physiology ; Retinal Ganglion Cells/metabolism ; Retinal Neoplasms/metabolism ; Retinoblastoma/metabolism ; Retinoblastoma Binding Proteins/*metabolism/physiology ; Ubiquitin-Protein Ligases/*metabolism/physiology ; },
abstract = {Three-dimensional (3D) organoid models derived from human pluripotent stem cells provide a platform for studying human development and understanding disease mechanisms. Most studies that examine biallelic inactivation of the cell cycle regulator Retinoblastoma 1 (RB1) and the link to retinoblastoma is in mice, however, less is known regarding the pathophysiological role of RB1 during human retinal development. To study the role of RB1 in early human retinal development and tumor formation, we generated retinal organoids from CRISPR/Cas9-derived RB1-null human embryonic stem cells (hESCs). We showed that RB is abundantly expressed in retinal progenitor cells in retinal organoids and loss of RB1 promotes S-phase entry. Furthermore, loss of RB1 resulted in widespread apoptosis and reduced the number of photoreceptor, ganglion, and bipolar cells. Interestingly, RB1 mutation in retinal organoids did not result in retinoblastoma formation in vitro or in the vitreous body of NOD/SCID immunodeficient mice. Together, our work identifies a crucial function for RB1 in human retinal development and suggests that RB1 deletion alone is not sufficient for tumor development, at least in human retinal organoids.},
}
@article {pmid32013399,
year = {2020},
author = {Zhang, D and Yan, Y and Que, H and Yang, T and Cheng, X and Ding, S and Zhang, X and Cheng, W},
title = {CRISPR/Cas12a-Mediated Interfacial Cleaving of Hairpin DNA Reporter for Electrochemical Nucleic Acid Sensing.},
journal = {ACS sensors},
volume = {5},
number = {2},
pages = {557-562},
doi = {10.1021/acssensors.9b02461},
pmid = {32013399},
issn = {2379-3694},
mesh = {CRISPR-Cas Systems/*genetics ; DNA/*chemistry ; Electrochemistry/*methods ; Humans ; Nucleic Acids/*chemistry ; },
abstract = {A rapid and sensitive isothermal method is crucial for point-of-care (POC) nucleic acid testing. Recently, RNA-guided CRISPR/Cas12a proteins were discovered to exhibit target-triggered nonspecific single-stranded deoxyribonuclease (ssDNase) activity. Herein, the ssDNase cleavage capacity of the CRISPR/Cas12a system for interfacial hairpin DNA (hpDNA) and linear DNA was investigated in detailed. A novel electrochemical DNA biosensor was then developed via target-induced Cas12a cleaving interfacial hpDNA. In this strategy, the RNA-guided target DNA binding activates the robust Cas12a ssDNase activity. The immobilized hpDNA electrochemical reporters with a low surface coverage and incompact morphological structure present accessible substrates for highly efficient Cas12a cleavage, leading to a highly sensitive electrochemical DNA biosensor. Under the optimal conditions, as low as 30 pM target DNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 nM. Furthermore, the practical application ability of the established sensing method for detecting the target in complex matrices was also demonstrated. The proposed strategy enables rapid and sensitive DNA determination, providing a potential tool for POC molecular diagnostics.},
}
@article {pmid31930919,
year = {2020},
author = {Yang, YB and Tang, YD and Hu, Y and Yu, F and Xiong, JY and Sun, MX and Lyu, C and Peng, JM and Tian, ZJ and Cai, XH and An, TQ},
title = {Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.},
journal = {Nano letters},
volume = {20},
number = {2},
pages = {1417-1427},
doi = {10.1021/acs.nanolett.9b05103},
pmid = {31930919},
issn = {1530-6992},
mesh = {CRISPR-Cas Systems/*genetics ; Capsid ; HeLa Cells ; Herpesvirus 1, Suid/*isolation &amp; purification/ultrastructure ; Humans ; Quantum Dots/*chemistry ; Virion/genetics/*isolation &amp; purification ; },
abstract = {Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.},
}
@article {pmid31465786,
year = {2020},
author = {Sen, P and Ghosal, S and Hazra, R and Arega, S and Mohanty, R and Kulkarni, KK and Budhwar, R and Ganguly, N},
title = {Transcriptomic analyses of gene expression by CRISPR knockout of miR-214 in cervical cancer cells.},
journal = {Genomics},
volume = {112},
number = {2},
pages = {1490-1499},
doi = {10.1016/j.ygeno.2019.08.020},
pmid = {31465786},
issn = {1089-8646},
mesh = {Biomarkers, Tumor/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; MicroRNAs/*genetics/metabolism ; *Transcriptome ; Uterine Cervical Neoplasms/*genetics ; },
abstract = {In this study, we investigate the effect of one such micro RNA, miR-214 which is frequently down-regulated in cervical cancer. In this study, we either CRISPR knocked out or overexpressed miR-214 in cervical cancer cells and analyzed the global mRNA expression by Next Generation Sequencing (NGS) It was observed that a total of 108 genes were upregulated and 178 downregulated between the samples, above and below the baseline respectively. Gene Ontology and KEGG pathway analysis reveal distinct biological processes and pathways. Analysis of gene regulatory networks also gave different network patterns in the two samples. We confirmed the RNA sequencing data for 10 genes; IFIF27, SMAD3, COX11, TP53INP1, ABL2, FGF8, TNFAIP3, NRG1, SP3 and MDM4 by Real-time PCR. This is the first report on the effect of miR-214 on global mRNA profile in cervical cancer cells. This study also reports new biomarkers for cervical cancer prognosis.},
}
@article {pmid33389894,
year = {2020},
author = {Chinnapaiyan, S and Dutta, RK and Unwalla, HJ},
title = {Novel Approaches of CRISPR-Cas Technology in Airway Diseases.},
journal = {Critical reviews in biomedical engineering},
volume = {48},
number = {3},
pages = {169-176},
doi = {10.1615/CritRevBiomedEng.2020034594},
pmid = {33389894},
issn = {1943-619X},
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR) technique plays a vital role in preclinical modelling of many respiratory diseases. Diseases such as chronic obstructive pulmonary disease (COPD), asthma, acute tracheal bronchitis, pneumonia, tuberculosis, lung cancer, and influenza infection continue to significantly impact human health. CRISPR associated (Cas) proteins, isolated from the immune system of prokaryotes, are one component of a very useful technique to manipulate gene sequences or editing and gene expression with significant implications for respiratory research in the field of molecular biology. CRISPR technology is a promising tool that is easily adaptable for specific editing of DNA sequences of interest with a goal towards modifying or eliminating gene function. Among its many potential applications, CRISPR can be applied to correcting genetic defects as well as for therapeutic approaches for treatment. This review elucidates recent advances in CRISPR-Cas technology in airway diseases.},
}
@article {pmid33389353,
year = {2021},
author = {Zhang, DF and Cui, XW and Li, WJ and Zhang, XM and Xue, HP and Huang, JK and Zhang, AH},
title = {Description of Salinimonas profundi sp. nov., a deep-sea bacterium harboring a transposon Tn6333.},
journal = {Antonie van Leeuwenhoek},
volume = {},
number = {},
pages = {},
pmid = {33389353},
issn = {1572-9699},
support = {31900001//National Natural Science Foundation of China/ ; 2017FF116(-002)//Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Yunnan University of Chinese Medicine/ ; 2019B02014//Fundamental Research Funds for the Central Universities/ ; },
abstract = {A Gram-staining-negative bacterium, strain HHU 13199T, was isolated from a marine sediment sample collected from South China Sea (119°19.896'E, 19°41.569'N) at a depth of 2918 m. The 16S rRNA gene sequence analysis indicated that strain HHU 13199T represents a member of the genus Salinimonas with the highest sequence similarity (99.8%) to the type strain S. iocasae KX18D6T. However, the average nucleotide identity values and digital DNA-DNA hybridization between strain HHU 13199T and closely related members of the genus Salinimonas were all below the cut-off level (95-96 % and 70%, respectively) for species delineation. This strain grew with sea salt of 0.5-18% (w/v) (optimum, 2-5%), but no growth observed when using NaCl instead. The major fatty acids are C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The predominant isoprenoid quinone is ubiquinone-8. The polar lipids mainly consist of phosphatidylethanolamine, and phosphatidylglycerol. Genomic characterization revealed that strain HHU 13199T harbors a distinct type I-F CRISPR-Cas system and plenty of genes associated with heavy metal resistance, including a transposon (Tn6333) conferring mercury resistance. In addition, a phylogenetic tree based on the bac120 core genes suggested that the genus Salinimonas should be a subclade within Alteromonas. On the basis of the phenotypic, phylogenetic and chemotaxonomic characterizations, strain HHU 13199T represents a novel species of the genus Salinimonas, for which the name Salinimonas profundi sp. nov. is proposed. The type strain is HHU 13199T (= KCTC 72837T = MCCC 1K04127T).},
}
@article {pmid33388855,
year = {2021},
author = {Chuang, YF and Phipps, AJ and Lin, FL and Hecht, V and Hewitt, AW and Wang, PY and Liu, GS},
title = {Approach for in vivo delivery of CRISPR/Cas system: a recent update and future prospect.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {},
number = {},
pages = {},
pmid = {33388855},
issn = {1420-9071},
support = {1185600//National Health and Medical Research Council/ ; 1123329//National Health and Medical Research Council/ ; ZDSYS20190902093409851//Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation/ ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system provides a groundbreaking genetic technology that allows scientists to modify genes by targeting specific genomic sites. Due to the relative simplicity and versatility of the CRISPR/Cas system, it has been extensively applied in human genetic research as well as in agricultural applications, such as improving crops. Since the gene editing activity of the CRISPR/Cas system largely depends on the efficiency of introducing the system into cells or tissues, an efficient and specific delivery system is critical for applying CRISPR/Cas technology. However, there are still some hurdles remaining for the translatability of CRISPR/Cas system. In this review, we summarized the approaches used for the delivery of the CRISPR/Cas system in mammals, plants, and aquacultures. We further discussed the aspects of delivery that can be improved to elevate the potential for CRISPR/Cas translatability.},
}
@article {pmid33385410,
year = {2020},
author = {Ghaemi, A and Bagheri, E and Abnous, K and Taghdisi, SM and Ramezani, M and Alibolandi, M},
title = {CRISPR-cas9 genome editing delivery systems for targeted cancer therapy.},
journal = {Life sciences},
volume = {},
number = {},
pages = {118969},
doi = {10.1016/j.lfs.2020.118969},
pmid = {33385410},
issn = {1879-0631},
abstract = {The prokaryotic CRISPR-Cas systems could be applied as revolutionized genome editing tool in live cells of various species to modify, visualize and identify definite sequences of DNA and RNA. CRISPR-Cas could edit the genome by homology-directed repair and non-homologous end joining mechanisms. Furthermore, DNA-targeting modification by CRISPR-Cas methodology provides opportunity for diagnosis, therapy and the genetic disorders investigation. Here, we summarized delivery systems employed for CRISPR-Cas9 for genome editing. Then preclinical studies of the CRISPR-Cas9-based therapeutics will be discussed considering the associated challenges and developments in its translation to clinic for cancer therapy.},
}
@article {pmid33383400,
year = {2020},
author = {Capdeville, N and Merker, L and Schindele, P and Puchta, H},
title = {Sophisticated CRISPR/Cas tools for fine-tuning plant performance.},
journal = {Journal of plant physiology},
volume = {257},
number = {},
pages = {153332},
doi = {10.1016/j.jplph.2020.153332},
pmid = {33383400},
issn = {1618-1328},
abstract = {Over the last years, the discovery of various natural and the development of a row of engineered CRISPR/Cas nucleases have made almost every site of plant genomes accessible for the induction of specific changes. Newly developed tools open up a wide range of possibilities for the induction of genetic variability, from changing a single bp to Mbps, and thus to fine-tune plant performance. Whereas early approaches focused on targeted mutagenesis, recently developed tools enable the induction of precise and predefined genomic modifications. The use of base editors allows the substitution of single nucleotides, whereas the use of prime editors and gene targeting methods enables the induction of larger sequence modifications from a few bases to several kbp. Recently, through CRISPR/Cas-mediated chromosome engineering, it became possible to induce heritable inversions and translocations in the Mbp range. Thus, a novel way of breaking and fixing genetic linkages has come into reach for breeders. In addition, sequence-specific recruitment of various factors involved in transcriptional and post-transcriptional regulation has been shown to provide an additional class of methods for the fine tuning of plant performance. In this review, we provide an overview of the most recent progress in the field of CRISPR/Cas-based tool development for plant genome engineering and try to evaluate the importance of these developments for breeding and biotechnological applications.},
}
@article {pmid33381141,
year = {2020},
author = {Peng, C and Zheng, M and Ding, L and Chen, X and Wang, X and Feng, X and Wang, J and Xu, J},
title = {Accurate Detection and Evaluation of the Gene-Editing Frequency in Plants Using Droplet Digital PCR.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {610790},
pmid = {33381141},
issn = {1664-462X},
abstract = {Gene-editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been reported that detect mutations at targeted loci induced by the CRISPR/Cas system in different organisms, they are semiquantitative and have difficulty in the detection of mutants in processed food samples containing low initial concentrations of DNA and may not accurately quantify editing frequency, especially at very low frequencies in a complex polyploid plant genome. In this study, we developed a duplexed dPCR-based method for the detection and evaluation of gene-editing frequencies in plants. We described the design, performance, accurate quantification, and comparison with other detection systems. The results show that the dPCR-based method is sensitive to different kinds of gene-editing mutations induced by gene-editing. Moreover, the method is applicable to polyploid plants and processed food samples containing low initial concentrations of DNA. Compared with qPCR and NGS-based methods, the dPCR method has a lower limit of detection (LOD) of the editing frequency and a better relationship with the expected editing frequency in detecting the edited region of gene-edited rice samples. Taken together, the duplexed dPCR assay is accurate and precise, and it will be a powerful tool for the detection and evaluation of gene-editing frequencies in plants in gene-editing technology.},
}
@article {pmid33380218,
year = {2020},
author = {Seastedt, KP and Pruett, N and Hoang, CD},
title = {Mouse models for mesothelioma drug discovery and development.},
journal = {Expert opinion on drug discovery},
volume = {},
number = {},
pages = {1-12},
doi = {10.1080/17460441.2021.1867530},
pmid = {33380218},
issn = {1746-045X},
abstract = {INTRODUCTION: Mesothelioma is an aggressive mesothelial lining tumor. Available drug therapies include chemotherapeutic agents, targeted molecular therapies, and immune system modulators. Mouse models were instrumental in the discovery and evaluation of such therapies, but there is need for improved understanding of the role of inflammation, tumor heterogeneity, mechanisms of carcinogenesis, and the tumor microenvironment. Novel mouse models may provide new insights and drive drug therapy discovery that improves efficacy.<br><br>AREAS COVERED: This review concerns available mouse models for mesothelioma drug discovery and development including the advantages and disadvantages of each. Gaps in current knowledge of mesothelioma are highlighted, and future directions for mouse model research are considered.<br><br>EXPERT OPINION: Soon, CRISPR-Cas gene-editing will improve understanding of mesothelioma mechanisms foundational to the discovery and testing of efficacious therapeutic targets. There are at least two likely areas of upcoming methodology development. One is concerned with precise modeling of inflammation - is it a causal process whereby inflammatory signals contribute to tumor initiation, or is it a secondary passenger process driven by asbestos exposure effects? The other area of methods improvement regards the availability of humanized immunocompromised mice harboring patient-derived xenografts. Combining human tumors in an environment with human immune cells will enable rapid innovation in immuno-oncology therapeutics.},
}
@article {pmid33378675,
year = {2020},
author = {Covarrubias, S and Vollmers, AC and Capili, A and Boettcher, M and Shulkin, A and Correa, MR and Halasz, H and Robinson, EK and O'Briain, L and Vollmers, C and Blau, J and Katzman, S and McManus, MT and Carpenter, S},
title = {High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.},
journal = {Cell reports},
volume = {33},
number = {13},
pages = {108541},
doi = {10.1016/j.celrep.2020.108541},
pmid = {33378675},
issn = {2211-1247},
abstract = {Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies.},
}
@article {pmid33374633,
year = {2020},
author = {Holý, O and Parra-Flores, J and Lepuschitz, S and Alarcón-Lavín, MP and Cruz-Córdova, A and Xicohtencatl-Cortes, J and Mancilla-Rojano, J and Ruppitsch, W and Forsythe, S},
title = {Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk.},
journal = {Foods (Basel, Switzerland)},
volume = {10},
number = {1},
pages = {},
doi = {10.3390/foods10010020},
pmid = {33374633},
issn = {2304-8158},
support = {191450 4R, 195420/EF//Universidad del Bío-Bio/ ; 991100531/39//Research Support Foundation, Vaduz/ ; },
abstract = {Cronobacter spp. are opportunistic pathogens of the Enterobacteriaceae family. The organism causes infections in all age groups, but the most serious cases occur in outbreaks related to neonates with meningitis and necrotizing enterocolitis. The objective was to determine the in silico and in vitro putative virulence factors of six Cronobacter sakazakii strains isolated from powdered milk (PM) in the Czech Republic. Strains were identified by MALDI-TOF MS and whole-genome sequencing (WGS). Virulence and resistance genes were detected with the Ridom SeqSphere+ software task template and the Comprehensive Antibiotic Resistance Database (CARD) platform. Adherence and invasion ability were performed using the mouse neuroblastoma (N1E-115 ATCCCRL-2263) cell line. The CRISPR-Cas system was searched with CRISPRCasFinder. Core genome MLST identified four different sequence types (ST1, ST145, ST245, and ST297) in six isolates. Strains 13755-1B and 1847 were able to adhere in 2.2 and 3.2 × 106 CFU/mL, while 0.00073% invasion frequency was detected only in strain 1847. Both strains 13755-1B and 1847 were positive for three (50.0%) and four virulence genes, respectively. The cpa gene was not detected. Twenty-eight genes were detected by WGS and grouped as flagellar or outer membrane proteins, chemotaxis, hemolysins, and invasion, plasminogen activator, colonization, transcriptional regulator, and survival in macrophages. The colistin-resistance-encoding mcr-9.1 and cephalothin-resis-encoding blaCSA genes and IncFII(pECLA) and IncFIB(pCTU3) plasmids were detected. All strains exhibited CRISPR matrices and four of them two type I-E and I-F matrices. Combined molecular methodologies improve Cronobacter spp. decision-making for health authorities to protect the population.},
}
@article {pmid33371847,
year = {2020},
author = {Qadri, H and Haseeb, A and Mir, M},
title = {Novel Strategiesto Combat the Emerging Drug Resistance in Human Pathogenic Microbes.},
journal = {Current drug targets},
volume = {},
number = {},
pages = {},
doi = {10.2174/1389450121666201228123212},
pmid = {33371847},
issn = {1873-5592},
abstract = {The major health-care burden for the developing world are the Infectious diseases and antimicrobial agents prove to be the magical drugs to combat this. But the phenomenon of antimicrobial resistance (AMR) represents a global challenging issue, which requires to be addressed effectively. The antimicrobial treatment for the emerging multidrug-resistant bacterial (e.g. TB, Cholera) and fungal (e.g. Candidiasis) infections is very limited and there are multiple causes and reasons responsible for the evolution of such resistance. Considering the critical issues of increasing AMR, there is an urgent requirement of identification, development, validation, and progression of novel strategies and approaches that can easily be utilized for overcoming this serious issue. Immunotherapy represents a significant way to improve host defenses and combat the issue of antimicrobial drug resistance. Similarly, drug combination therapy represents another promising approach for reducing the evolution of resistance and enhancing the longevity of the antimicrobial agents. Bacteriophage therapy also acts as a novel therapeutic option to control the development of the multidrug resistance (MDR) phenomenon. Besides, CRISPR, an innovative genome editing technology offers multiple applications to safeguard host defenses to overcome different resistance challenges. The novel approaches/strategies like combination therapy, bacteriophage therapy, immunotherapy, and CRISPR/Cas discussed here presents an overview of some of the novel strategies/approaches to be adopted against the pathogenic microbes/microbial invasions along with advanced knowledge of different drug resistance mechanisms adopted by the microbial pathogens to gain resistance against different antimicrobial agents. Therefore, understanding the novel control plans/approaches and different drug resistance mechanisms will help achieve the goals of the successful development of potential antimicrobial drugs and their respective targets and eventually help curtail the problem of increasing antimicrobial drug resistance menace in various human pathogenic microbes.},
}
@article {pmid33371215,
year = {2020},
author = {Lyu, P and Wang, L and Lu, B},
title = {Virus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing.},
journal = {Life (Basel, Switzerland)},
volume = {10},
number = {12},
pages = {},
pmid = {33371215},
issn = {2075-1729},
support = {W81XWH2010265//DOD/ ; },
abstract = {The discovery of designer nucleases has made genome editing much more efficient than before. The designer nucleases have been widely used for mechanistic studies, animal model generation and gene therapy development. However, potential off-targets and host immune responses are issues still need to be addressed for in vivo uses, especially clinical applications. Short term expression of the designer nucleases is necessary to reduce both risks. Currently, various delivery methods are being developed for transient expression of designer nucleases including Zinc Finger Nuclease (ZNF), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas). Recently, virus-like particles are being used for gene editing. In this review, we will talk through commonly used genome editing nucleases, discuss gene editing delivery tools and review the latest literature using virus-like particles to deliver gene editing effectors.},
}
@article {pmid33369608,
year = {2020},
author = {Smith, A and Bergwell, M and Smith, E and Mathew, D and Iyer, J},
title = {CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {166},
pages = {},
doi = {10.3791/62001},
pmid = {33369608},
issn = {1940-087X},
abstract = {The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing in less than two weeks.},
}
@article {pmid33369120,
year = {2020},
author = {Zhan, X and Lu, Y and Zhu, JK and Botella, JR},
title = {Genome editing for plant research and crop improvement.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13063},
pmid = {33369120},
issn = {1744-7909},
abstract = {The advent of CRISPR has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V and VI systems for gene disruption as well as for precise sequence alterations, gene transcription and epigenome control. This article is protected by copyright. All rights reserved.},
}
@article {pmid33365307,
year = {2020},
author = {Lian, J and Wang, Y and Luo, Y and Li, C},
title = {Editorial: Development and Application of Novel Genome Engineering Tools in Microbial Biotechnology.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {621851},
doi = {10.3389/fbioe.2020.621851},
pmid = {33365307},
issn = {2296-4185},
}
@article {pmid33363552,
year = {2020},
author = {Deguchi, M and Kane, S and Potlakayala, S and George, H and Proano, R and Sheri, V and Curtis, WR and Rudrabhatla, S},
title = {Metabolic Engineering Strategies of Industrial Hemp (Cannabis sativa L.): A Brief Review of the Advances and Challenges.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {580621},
pmid = {33363552},
issn = {1664-462X},
abstract = {Industrial hemp (Cannabis sativa L.) is a diploid (2n = 20), dioecious plant that is grown for fiber, seed, and oil. Recently, there has been a renewed interest in this crop because of its panoply of cannabinoids, terpenes, and other phenolic compounds. Specifically, hemp contains terpenophenolic compounds such as cannabidiol (CBD) and cannabigerol (CBG), which act on cannabinoid receptors and positively regulate various human metabolic, immunological, and physiological functions. CBD and CBG have an effect on the cytokine metabolism, which has led to the examination of cannabinoids on the treatment of viral diseases, including COVID-19. Based on genomic, transcriptomic, and metabolomic studies, several synthetic pathways of hemp secondary metabolite production have been elucidated. Nevertheless, there are few reports on hemp metabolic engineering despite obvious impact on scientific and industrial sectors. In this article, recent status and current perspectives on hemp metabolic engineering are reviewed. Three distinct approaches to expedite phytochemical yield are discussed. Special emphasis has been placed on transgenic and transient gene delivery systems, which are critical for successful metabolic engineering of hemp. The advent of new tools in synthetic biology, particularly the CRISPR/Cas systems, enables environment-friendly metabolic engineering to increase the production of desirable hemp phytochemicals while eliminating the psychoactive compounds, such as tetrahydrocannabinol (THC).},
}
@article {pmid33363214,
year = {2020},
author = {Yao, L and Zhang, Q and Li, A and Ma, B and Zhang, Z and Liu, J and Liang, L and Zhu, S and Gan, Y and Zhang, Q},
title = {Synthetic Artificial Long Non-coding RNA Shows Higher Efficiency in Specific Malignant Phenotype Inhibition Compared to the CRISPR/Cas Systems.},
journal = {Frontiers in molecular biosciences},
volume = {7},
number = {},
pages = {617600},
pmid = {33363214},
issn = {2296-889X},
abstract = {Objective: Both oncogenic transcription factors (TFs) and microRNAs (miRNAs) play an important regulator in human cancer by transcriptional and post-transcriptional regulation, respectively. These phenomena raise questions about the ability of artificial device to regulate miRNAs and TFs simultaneously. In this study, we aimed to construct an artificial long non-coding RNA, &quot;alncRNA,&quot; which imitated CRISPR/Cas systems and to illuminate its therapeutic effects in bladder cancer cell lines. At the same time, we also compared the efficiency of alncRNA and CRISPR/Cas systems in regulating gene expression. Study Design and Methods: Based on engineering principles of synthetic biology, we combined tandem arrayed cDNA sequences of aptamer for TFs with tandem arrayed cDNA copies of binding sites for the miRNAs to construct alncRNA. In order to prove the utility of this platform, we chose β -catenin, NF-κB, miR-940, and miR-495 as the functional targets and used the bladder cancer cell lines 5637 and T24 as the test models. Real-time Quantitative PCR (qPCR), dual-luciferase assay and relative phenotypic experiments were applied to severally test the expression of relative gene and therapeutic effects of our devices. Result: Dual-luciferase assay indicated alncRNA could inhibit transcriptional activity of TFs. What's more, the result of qPCR showed that expression levels of the relative TFs target genes and miRNAs were reduced by corresponding alncRNA and the inhibitory effect was better than CRIPSR dCas9-KRAB. By functional experiments, decreased cell proliferation, increased apoptosis, and motility inhibition were observed in alncRNA-infected bladder cells. Conclusion: In summary, our synthetic devices indeed function as anti-tumor regulator, which synchronously accomplish transcriptional and post-transcriptional regulation in bladder cancer cell and show higher efficiency in specific malignant phenotype inhibition compared to the CRISPR/Cas systems. Most importantly, Anti-cancer effects were induced by the synthetic alncRNA in the bladder cancer lines. Our devices, therefore, provides a novel strategy for cancer therapy and could be a useful &quot;weapon&quot; for cancer cell.},
}
@article {pmid33362853,
year = {2020},
author = {Zhang, N and Bewick, B and Xia, G and Furling, D and Ashizawa, T},
title = {A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1.},
journal = {Frontiers in genetics},
volume = {11},
number = {},
pages = {594576},
pmid = {33362853},
issn = {1664-8021},
abstract = {Cas13a, an effector of type VI CRISPR-Cas systems, is an RNA guided RNase with multiplexing and therapeutic potential. This study employs the Leptotrichia shahii (Lsh) Cas13a and a repeat-based CRISPR RNA (crRNA) to track and eliminate toxic RNA aggregates in myotonic dystrophy type 1 (DM1) - a neuromuscular disease caused by CTG expansion in the DMPK gene. We demonstrate that LshCas13a cleaves CUG repeat RNA in biochemical assays and reduces toxic RNA load in patient-derived myoblasts. As a result, LshCas13a reverses the characteristic adult-to-embryonic missplicing events in several key genes that contribute to DM1 phenotype. The deactivated LshCas13a can further be repurposed to track RNA-rich organelles within cells. Our data highlights the reprogrammability of LshCas13a and the possible use of Cas13a to target expanded repeat sequences in microsatellite expansion diseases.},
}
@article {pmid33361628,
year = {2020},
author = {Kaul, T and Sony, SK and Verma, R and Motelb, KFA and Prakash, AT and Eswaran, M and Bharti, J and Nehra, M and Kaul, R},
title = {Revisiting CRISPR/Cas-mediated crop improvement: Special focus on nutrition.},
journal = {Journal of biosciences},
volume = {45},
number = {},
pages = {},
pmid = {33361628},
issn = {0973-7138},
abstract = {Genome editing (GE) technology has emerged as a multifaceted strategy that instantaneously popularised the mechanism to modify the genetic constitution of an organism. The clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein-based genome editing (CRISPR/Cas) approach has huge potential for efficacious editing of genomes of numerous organisms. This framework has demonstrated to be more economical in contrast to mega-nucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) for its flexibility, versatility, and potency. The advent of sequence-specific nucleases (SSNs) allowed the precise induction of double-strand breaks (DSBs) into the genome, ensuring desired alterations through non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. Researchers have utilized CRISPR/Cas-mediated genome alterations across crop varieties to generate desirable characteristics for yield enhancement, enriched nutritional quality, and stressresistance. Here, we highlighted the recent progress in the area of nutritional improvement of crops via the CRISPR/Cas-based tools for fundamental plant research and crop genetic advancements. Application of this genome editing aids in unraveling the basic biology facts in plants supplemented by the incorporation of genome-wide association studies, artificial intelligence, and various bioinformatic frameworks, thereby providing futuristic model studies and their affirmations. Strategies for reducing the 'off-target' effects and the societal approval of genome-modified crops developed via this modern biotechnological approach have been reviewed.},
}
@article {pmid33360715,
year = {2020},
author = {Fage, C and Lemire, N and Moineau, S},
title = {Delivery of CRISPR-Cas systems using phage-based vectors.},
journal = {Current opinion in biotechnology},
volume = {68},
number = {},
pages = {174-180},
doi = {10.1016/j.copbio.2020.11.012},
pmid = {33360715},
issn = {1879-0429},
abstract = {Antimicrobial resistance has spread quickly on a worldwide scale, reducing therapeutic options for bacterial infections. CRISPR-Cas is an adaptive immune system found in many prokaryotes that can be designed to target bacterial genomes, leading to cell death. Repurposing the CRISPR-Cas system as a therapeutic strategy offers an attractive way to overcome antimicrobial resistance. However, this strategy requires efficient vectors for the CRISPR-Cas system to reach the bacterial genomes. Engineered phages offer an attractive option as cargo delivery vectors. In this review, we discuss the production of phage-based vectors and the relevance of using repurposed CRISPR-Cas systems as antimicrobials. We also discuss recent progress in phage engineering that can potentially overcome the limitations and increase the efficiency of CRISPR-Cas delivery.},
}
@article {pmid33360689,
year = {2020},
author = {Jandova, J and Wondrak, GT},
title = {Genomic GLO1 deletion modulates TXNIP expression, glucose metabolism, and redox homeostasis while accelerating human A375 malignant melanoma tumor growth.},
journal = {Redox biology},
volume = {39},
number = {},
pages = {101838},
pmid = {33360689},
issn = {2213-2317},
abstract = {Glyoxalase 1 (encoded by GLO1) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human malignant melanoma cells and patient tumors and substantiated a novel role of GLO1 as a molecular determinant of invasion and metastasis in melanoma. Here, employing NanoString™ gene expression profiling (nCounter™ 'PanCancer Progression Panel'), we report that CRISPR/Cas 9-based GLO1 deletion from human A375 malignant melanoma cells alters glucose metabolism and redox homeostasis, observable together with acceleration of tumorigenesis. Nanostring™ analysis identified TXNIP (encoding thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis, displaying the most pronounced expression change in response to GLO1 elimination, confirmed by RT-qPCR and immunoblot analysis. TXNIP was also upregulated in CRISPR/Cas9-engineered DU145 prostate carcinoma cells lacking GLO1, and treatment with MG or a pharmacological GLO1 inhibitor (TLSC702) mimicked GLO1_KO status, suggesting that GLO1 controls TXNIP expression through regulation of MG. GLO1_KO status was characterized by (i) altered oxidative stress response gene expression, (ii) attenuation of glucose uptake and metabolism with downregulation of gene expression (GLUT1, GFAT1, GFAT2, LDHA) and depletion of related key metabolites (glucose-6-phosphate, UDP-N-acetylglucosamine), and (iii) immune checkpoint modulation (PDL1). While confirming our earlier finding that GLO1 deletion limits invasion and metastasis with modulation of EMT-related genes (e.g. TGFBI, MMP9, ANGPTL4, TLR4, SERPINF1), we observed that GLO1_KO melanoma cells displayed a shortened population doubling time, cell cycle alteration with increased M-phase population, and enhanced anchorage-independent growth, a phenotype supported by expression analysis (CXCL8, CD24, IL1A, CDKN1A). Concordantly, an accelerated growth rate of GLO1_KO tumors, accompanied by TXNIP overexpression and metabolic reprogramming, was observable in a SCID mouse melanoma xenograft model, demonstrating that A375 melanoma tumor growth and metastasis can be dysregulated in opposing ways as a consequence of GLO1 elimination.},
}
@article {pmid33358112,
year = {2020},
author = {Peng, R and Zhang, B},
title = {Foxtail Millet: A New Model for C4 Plants.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2020.12.003},
pmid = {33358112},
issn = {1878-4372},
abstract = {Arabidopsis and rice are major models for C3 plants, but we still lack a model for C4 plants. Recently, Yang and coworkers developed foxtail millet as a C4 plant model; with the rapid development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technology, this will open a new era for plant functional studies and crop improvement.},
}
@article {pmid33356428,
year = {2020},
author = {Cui, X and Liu, K and Atta, S and Zeng, C and Zhou, CY and Wang, X},
title = {Two unique prophages of &quot;Candidatus Liberibacter asiaticus&quot; strains from Pakistan.},
journal = {Phytopathology},
volume = {},
number = {},
pages = {},
doi = {10.1094/PHYTO-10-20-0454-SC},
pmid = {33356428},
issn = {0031-949X},
abstract = {&quot;Candidatus Liberibacter asiaticus&quot; (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing (NGS) analysis. P-PA19-1 prophage has high sequence similarity (Identity: 78.23%) at the early-gene region of prophage SC1 (Type 1) but it is significantly divergent in the late-gene region (Identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (Identity: 97.96%), and also in the early gene region except for a deletion of a 7,179 bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.},
}
@article {pmid33356427,
year = {2020},
author = {Wheatley, M and Yang, Y},
title = {Versatile Applications of the CRISPR/Cas Toolkit in Plant Pathology and Disease Management.},
journal = {Phytopathology},
volume = {},
number = {},
pages = {},
doi = {10.1094/PHYTO-08-20-0322-IA},
pmid = {33356427},
issn = {0031-949X},
abstract = {New tools and advanced technologies have played key roles in facilitating basic research in plant pathology and practical approaches for disease management and crop health. Recently, the CRISPR/Cas (clustered regularly interspersed short palindromic repeats/CRISPR associated) system has emerged as a powerful and versatile tool for genome editing and other molecular applications. This review aims to introduce and highlight the CRISPR/Cas toolkit and its current and future impact on plant pathology and disease management. We will cover the rapidly expanding horizon of various CRISPR/Cas applications in the basic study of plant-pathogen interactions, genome engineering of plant disease resistance, and molecular diagnosis of diverse pathogens. Using the citrus greening disease as an example, various CRISPR/Cas-enabled strategies are presented to precisely edit the host genome for disease resistance, to rapidly detect the pathogen for disease management, and to potentially use gene drive for insect population control. At the cutting edge of nucleic acid manipulation and detection, the CRISPR/Cas toolkit will accelerate plant breeding and reshape crop production and disease management as we face the challenges in 21st century agriculture.},
}
@article {pmid33353186,
year = {2020},
author = {Galow, AM and Goldammer, T and Hoeflich, A},
title = {Xenogeneic and Stem Cell-Based Therapy for Cardiovascular Diseases: Genetic Engineering of Porcine Cells and Their Applications in Heart Regeneration.},
journal = {International journal of molecular sciences},
volume = {21},
number = {24},
pages = {},
pmid = {33353186},
issn = {1422-0067},
support = {ESF /14-BM-A55-0028/18//EU Social Fund/ ; },
abstract = {Cardiovascular diseases represent a major health concern worldwide with few therapy options for ischemic injuries due to the limited regeneration potential of affected cardiomyocytes. Innovative cell replacement approaches could facilitate efficient regenerative therapy. However, despite extensive attempts to expand primary human cells in vitro, present technological limitations and the lack of human donors have so far prevented their broad clinical use. Cell xenotransplantation might provide an ethically acceptable unlimited source for cell replacement therapies and bridge the gap between waiting recipients and available donors. Pigs are considered the most suitable candidates as a source for xenogeneic cells and tissues due to their anatomical and physiological similarities with humans. The potential of porcine cells in the field of stem cell-based therapy and regenerative medicine is under intensive investigation. This review outlines the current progress and highlights the most promising approaches in xenogeneic cell therapy with a focus on the cardiovascular system.},
}
@article {pmid33353099,
year = {2020},
author = {Kim, D and Le, QV and Wu, Y and Park, J and Oh, YK},
title = {Nanovesicle-Mediated Delivery Systems for CRISPR/Cas Genome Editing.},
journal = {Pharmaceutics},
volume = {12},
number = {12},
pages = {},
pmid = {33353099},
issn = {1999-4923},
support = {NRF-2018R1A2A1A05019203//Ministry of Science and ICT/ ; NRF-2018R1A5A2024425//Ministry of Science and ICT/ ; HI15C2842//Ministry of Health &amp; Welfare/ ; HI18C2177//Ministry of Health &amp; Welfare/ ; },
abstract = {Genome-editing technology has emerged as a potential tool for treating incurable diseases for which few therapeutic modalities are available. In particular, discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system together with the design of single-guide RNAs (sgRNAs) has sparked medical applications of genome editing. Despite the great promise of the CRISPR/Cas system, its clinical application is limited, in large part, by the lack of adequate delivery technology. To overcome this limitation, researchers have investigated various systems, including viral and nonviral vectors, for delivery of CRISPR/Cas and sgRNA into cells. Among nonviral delivery systems that have been studied are nanovesicles based on lipids, polymers, peptides, and extracellular vesicles. These nanovesicles have been designed to increase the delivery of CRISPR/Cas and sgRNA through endosome escape or using various stimuli such as light, pH, and environmental features. This review covers the latest research trends in nonviral, nanovesicle-based delivery systems that are being applied to genome-editing technology and suggests directions for future progress.},
}
@article {pmid33352158,
year = {2020},
author = {Zhang, S and Li, T and Huo, Y and Yang, J and Fleming, J and Shi, M and Wang, Y and Wei, W and Gu, S and Bi, L and Jiang, T and Zhang, H},
title = {Mycobacterium tuberculosis CRISPR/Cas system Csm1 holds clues to the evolutionary relationship between DNA polymerase and cyclase activity.},
journal = {International journal of biological macromolecules},
volume = {170},
number = {},
pages = {140-149},
doi = {10.1016/j.ijbiomac.2020.12.014},
pmid = {33352158},
issn = {1879-0003},
abstract = {Prokaryotic CRISPR/Cas systems confer immunity against invading nucleic acids through effector complexes. Csm1, the signature protein of Type III effector complexes, catalyses cyclic oligoadenylate synthesis when in the effector complex, but not when alone, activating the Csm6 nuclease and switching on the antiviral response. Here, we provide biochemical evidence that M. tuberculosis Csm1 (MtbCsm1) has ion-dependent polymerase activity when independent of the effector complex. Structural studies provide supporting evidence that the catalytic core of the MtbCsm1 palm2 domain is almost identical to that of classical DNA polymerase Pol IV, and that the palm1 and B domains function as the other structural elements required (thumb and fingers) for DNA polymerase activity. MtbCsm1 polymerase activity is relatively weak in vitro and its functional relevance in vivo is unknown. Our structural and mutagenesis data suggest that residue K692 in the palm2 domain has been significant in the evolution of Csm1 from a polymerase to a cyclase, and support the notion that the cyclase activity of Csm1 requires the presence of other elements provided by the CRISPR/Cas effector complex. This structural rationale for Csm1 polymerase (alone) and cyclase (within the effector complex) activity should benefit future functional investigations and engineering.},
}
@article {pmid33349652,
year = {2020},
author = {Wheatley, RM and MacLean, RC},
title = {CRISPR-Cas systems restrict horizontal gene transfer in Pseudomonas aeruginosa.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
pmid = {33349652},
issn = {1751-7370},
abstract = {CRISPR-Cas systems provide bacteria and archaea with an adaptive immune system that targets foreign DNA. However, the xenogenic nature of immunity provided by CRISPR-Cas raises the possibility that these systems may constrain horizontal gene transfer. Here we test this hypothesis in the opportunistic pathogen Pseudomonas aeruginosa, which has emerged as an important model system for understanding CRISPR-Cas function. Across the diversity of P. aeruginosa, active CRISPR-Cas systems are associated with smaller genomes and higher GC content, suggesting that CRISPR-Cas inhibits the acquisition of foreign DNA. Although phage is the major target of CRISPR-Cas spacers, more than 80% of isolates with an active CRISPR-Cas system have spacers that target integrative conjugative elements (ICE) or the conserved conjugative transfer machinery used by plasmids and ICE. Consistent with these results, genomes containing active CRISPR-Cas systems harbour a lower abundance of both prophage and ICE. Crucially, spacers in genomes with active CRISPR-Cas systems map to ICE and phage that are integrated into the chromosomes of closely related genomes lacking CRISPR-Cas immunity. We propose that CRISPR-Cas acts as an important constraint to horizontal gene transfer, and the evolutionary mechanisms that ensure its maintenance or drive its loss are key to the ability of this pathogen to adapt to new niches and stressors.},
}
@article {pmid33349060,
year = {2020},
author = {Pillon, MC and Gordon, J and Frazier, MN and Stanley, RE},
title = {HEPN RNases - an emerging class of functionally distinct RNA processing and degradation enzymes.},
journal = {Critical reviews in biochemistry and molecular biology},
volume = {},
number = {},
pages = {1-30},
doi = {10.1080/10409238.2020.1856769},
pmid = {33349060},
issn = {1549-7798},
abstract = {HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) RNases are an emerging class of functionally diverse RNA processing and degradation enzymes. Members are defined by a small α-helical bundle encompassing a short consensus RNase motif. HEPN dimerization is a universal requirement for RNase activation as the conserved RNase motifs are precisely positioned at the dimer interface to form a composite catalytic center. While the core HEPN fold is conserved, the organization surrounding the HEPN dimer can support large structural deviations that contribute to their specialized functions. HEPN RNases are conserved throughout evolution and include bacterial HEPN RNases such as CRISPR-Cas and toxin-antitoxin associated nucleases, as well as eukaryotic HEPN RNases that adopt large multi-component machines. Here we summarize the canonical elements of the growing HEPN RNase family and identify molecular features that influence RNase function and regulation. We explore similarities and differences between members of the HEPN RNase family and describe the current mechanisms for HEPN RNase activation and inhibition.},
}
@article {pmid33346713,
year = {2020},
author = {Moreb, EA and Hutmacher, M and Lynch, MD},
title = {CRISPR-Cas &quot;Non-Target&quot; Sites Inhibit On-Target Cutting Rates.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {550-561},
doi = {10.1089/crispr.2020.0065},
pmid = {33346713},
issn = {2573-1602},
abstract = {CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. The number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.},
}
@article {pmid33346710,
year = {2020},
author = {Chaudhari, HG and Penterman, J and Whitton, HJ and Spencer, SJ and Flanagan, N and Lei Zhang, MC and Huang, E and Khedkar, AS and Toomey, JM and Shearer, CA and Needham, AW and Ho, TW and Kulman, JD and Cradick, TJ and Kernytsky, A},
title = {Evaluation of Homology-Independent CRISPR-Cas9 Off-Target Assessment Methods.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {440-453},
pmid = {33346710},
issn = {2573-1602},
abstract = {The ability to alter genomes specifically by CRISPR-Cas gene editing has revolutionized biological research, biotechnology, and medicine. Broad therapeutic application of this technology, however, will require thorough preclinical assessment of off-target editing by homology-based prediction coupled with reliable methods for detecting off-target editing. Several off-target site nomination assays exist, but careful comparison is needed to ascertain their relative strengths and weaknesses. In this study, HEK293T cells were treated with Streptococcus pyogenes Cas9 and eight guide RNAs with varying levels of predicted promiscuity in order to compare the performance of three homology-independent off-target nomination methods: the cell-based assay, GUIDE-seq, and the biochemical assays CIRCLE-seq and SITE-seq. The three methods were benchmarked by sequencing 75,000 homology-nominated sites using hybrid capture followed by high-throughput sequencing, providing the most comprehensive assessment of such methods to date. The three methods performed similarly in nominating sequence-confirmed off-target sites, but with large differences in the total number of sites nominated. When combined with homology-dependent nomination methods and confirmation by sequencing, all three off-target nomination methods provide a comprehensive assessment of off-target activity. GUIDE-seq's low false-positive rate and the high correlation of its signal with observed editing highlight its suitability for nominating off-target sites for ex vivo CRISPR-Cas therapies.},
}
@article {pmid33346707,
year = {2020},
author = {Shmakov, SA and Utkina, I and Wolf, YI and Makarova, KS and Severinov, KV and Koonin, EV},
title = {CRISPR Arrays Away from cas Genes.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {535-549},
pmid = {33346707},
issn = {2573-1602},
abstract = {CRISPR-Cas systems typically consist of a CRISPR array and cas genes that are organized in one or more operons. However, a substantial fraction of CRISPR arrays are not adjacent to cas genes. Definitive identification of such isolated CRISPR arrays runs into the problem of false-positives, with unrelated types of repetitive sequences mimicking CRISPR. We developed a computational pipeline to eliminate false CRISPR predictions and found that up to 25% of the CRISPR arrays in complete bacterial and archaeal genomes are located away from cas genes. Most of the repeats in these isolated arrays are identical to repeats in cas-adjacent CRISPR arrays in the same or closely related genomes, indicating an evolutionary relationship between isolated arrays and arrays in typical CRISPR-cas loci. The spacers in isolated CRISPR arrays show nearly as many matches to viral genomes as spacers from complete CRISPR-cas loci, suggesting that the isolated arrays were either functionally active recently or continue to function. Reconstruction of evolutionary events in closely related bacterial genomes suggests three routes of evolution of isolated CRISPR arrays: (1) loss of cas genes in a CRISPR-cas locus, (2) de novo generation of arrays from off-target spacer integration into sequences resembling the corresponding repeats, and (3) transfer by mobile genetic elements. Both combination of de novo emerging arrays with cas genes and regain of cas genes by isolated arrays via recombination likely contribute to functional diversification in CRISPR-Cas evolution.},
}
@article {pmid33346665,
year = {2020},
author = {Choi, JH and Lim, J and Shin, M and Paek, SH and Choi, JW},
title = {CRISPR-Cas12a-Based Nucleic Acid Amplification-Free DNA Biosensor via Au Nanoparticle-Assisted Metal-Enhanced Fluorescence and Colorimetric Analysis.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.0c04303},
pmid = {33346665},
issn = {1530-6992},
abstract = {Cell-free DNA (cfDNA) has attracted significant attention due to its high potential to diagnose diseases, such as cancer. Still, its detection by amplification method has limitations because of false-positive signals and difficulty in designing target-specific primers. CRISPR-Cas-based fluorescent biosensors have been developed but also need the amplification step for the detection. In this study, for the first time CRISPR-Cas12a based nucleic acid amplification-free fluorescent biosensor was developed to detect cfDNA by a metal-enhanced fluorescence (MEF) using DNA-functionalized Au nanoparticle (AuNP). Upon activating the CRISPR-Cas12a complex by the target cfDNA and subsequent single-strand DNA (ssDNA) degradation between AuNP and fluorophore, MEF occurred with color changes from purple to red-purple. Using this system, breast cancer gene-1 (BRCA-1) can be detected with very high sensitivity in 30 min. This rapid and highly selective sensor can be applied to measure other nucleic acid biomarkers such as viral DNA in field-deployable and point-of-care testing (POCT) platform.},
}
@article {pmid33343607,
year = {2020},
author = {Maioli, A and Gianoglio, S and Moglia, A and Acquadro, A and Valentino, D and Milani, AM and Prohens, J and Orzaez, D and Granell, A and Lanteri, S and Comino, C},
title = {Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {607161},
pmid = {33343607},
issn = {1664-462X},
abstract = {Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.},
}
@article {pmid33339441,
year = {2020},
author = {Janik, E and Niemcewicz, M and Ceremuga, M and Krzowski, L and Saluk-Bijak, J and Bijak, M},
title = {Various Aspects of a Gene Editing System-CRISPR-Cas9.},
journal = {International journal of molecular sciences},
volume = {21},
number = {24},
pages = {},
pmid = {33339441},
issn = {1422-0067},
abstract = {The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR-Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR-Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR-Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR-Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR-Cas9.},
}
@article {pmid33338493,
year = {2020},
author = {Mejdani, M and Pawluk, A and Maxwell, KL and Davidson, AR},
title = {Anti-CRISPR AcrIE2 binds the type I-E CRISPR-Cas complex but does not block DNA binding.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {166759},
doi = {10.1016/j.jmb.2020.166759},
pmid = {33338493},
issn = {1089-8638},
abstract = {Anti-CRISPRs are protein inhibitors of CRISPR-Cas systems. They are produced by phages and other mobile genetic elements to evade CRISPR-Cas-mediated destruction. Anti-CRISPRs are remarkably diverse in sequence, structure, and functional mechanism; thus, structural and mechanistic investigations of anti-CRISPRs continue to yield exciting new insights. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AcrIE2, an anti-CRISPR that inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. Guided by the structure, we used site-directed mutagenesis to identify key residues that are required for AcrIE2 function. Using affinity purification experiments, we found that AcrIE2 binds the type I-E CRISPR-Cas complex (Cascade). In vivo transcriptional assays, in which Cascade was targeted to promoter regions, demonstrated that Cascade still binds to DNA in the presence of AcrIE2. This is the first instance of a type I anti-CRISPR that binds to a CRISPR-Cas complex but does not prevent DNA-binding. Another unusual property of AcrIE2 is that the effect of Cascade:AcrIE2 complex binding to promoter regions varied depending on the position of the binding site. Most surprisingly, Cascade:AcrIE2 binding led to transcriptional activation in some cases rather than repression, which did not occur when Cascade alone bound to the same sites. We conclude that AcrIE2 operates through a distinct mechanism compared to other type I anti-CRISPRs. While AcrIE2 does not prevent Cascade from binding DNA, it likely blocks subsequent recruitment of the Cas3 nuclease to Cascade thereby preventing DNA cleavage.},
}
@article {pmid33335523,
year = {2020},
author = {Ahmad, S and Huang, Q and Ni, J and Xiao, Y and Yang, Y and Shen, Y},
title = {Functional Analysis of the NucS/EndoMS of the Hyperthermophilic Archaeon Sulfolobus islandicus REY15A.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {607431},
pmid = {33335523},
issn = {1664-302X},
abstract = {EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the in vitro mismatch cleavage activity and in vivo function of an EndoMS homolog (SisEndoMS) from Sulfolobus islandicus REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn2+ was used as the co-factor instead of Mg2+, SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆endoMS by counter selection against 5-fluoroorotic acid (5-FOA). The endoMS knockout mutant had much higher spontaneous mutation rate (5.06 × 10-3) than that of the wild type (4.6 × 10-6). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in S. islandicus resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in Sulfolobus which have a single chromosome is discussed.},
}
@article {pmid33334019,
year = {2020},
author = {Moranguinho, I and Valente, ST},
title = {Block-And-Lock: New Horizons for a Cure for HIV-1.},
journal = {Viruses},
volume = {12},
number = {12},
pages = {},
pmid = {33334019},
issn = {1999-4915},
support = {R01 AI118432/AI/NIAID NIH HHS/United States ; R01 AI097012/AI/NIAID NIH HHS/United States ; R61 AI140439/AI/NIAID NIH HHS/United States ; R33 AI116226/AI/NIAID NIH HHS/United States ; },
abstract = {HIV-1/AIDS remains a global public health problem. The world health organization (WHO) reported at the end of 2019 that 38 million people were living with HIV-1 worldwide, of which only 67% were accessing antiretroviral therapy (ART). Despite great success in the clinical management of HIV-1 infection, ART does not eliminate the virus from the host genome. Instead, HIV-1 remains latent as a viral reservoir in any tissue containing resting memory CD4+ T cells. The elimination of these residual proviruses that can reseed full-blown infection upon treatment interruption remains the major barrier towards curing HIV-1. Novel approaches have recently been developed to excise or disrupt the virus from the host cells (e.g., gene editing with the CRISPR-Cas system) to permanently shut off transcription of the virus (block-and-lock and RNA interference strategies), or to reactivate the virus from cell reservoirs so that it can be eliminated by the immune system or cytopathic effects (shock-and-kill strategy). Here, we will review each of these approaches, with the major focus placed on the block-and-lock strategy.},
}
@article {pmid33333349,
year = {2021},
author = {Chu, W and Prodromou, R and Day, KN and Schneible, JD and Bacon, KB and Bowen, JD and Kilgore, RE and Catella, CM and Moore, BD and Mabe, MD and Alashoor, K and Xu, Y and Xiao, Y and Menegatti, S},
title = {Peptides and pseudopeptide ligands: a powerful toolbox for the affinity purification of current and next-generation biotherapeutics.},
journal = {Journal of chromatography. A},
volume = {1635},
number = {},
pages = {461632},
doi = {10.1016/j.chroma.2020.461632},
pmid = {33333349},
issn = {1873-3778},
abstract = {Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as β-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.},
}
@article {pmid33333121,
year = {2020},
author = {Suzuki, Y and Onuma, H and Sato, R and Sato, Y and Hashiba, A and Maeki, M and Tokeshi, M and Kayesh, MEH and Kohara, M and Tsukiyama-Kohara, K and Harashima, H},
title = {Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {330},
number = {},
pages = {61-71},
doi = {10.1016/j.jconrel.2020.12.013},
pmid = {33333121},
issn = {1873-4995},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.},
}
@article {pmid33333018,
year = {2020},
author = {Takeda, SN and Nakagawa, R and Okazaki, S and Hirano, H and Kobayashi, K and Kusakizako, T and Nishizawa, T and Yamashita, K and Nishimasu, H and Nureki, O},
title = {Structure of the miniature type V-F CRISPR-Cas effector enzyme.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2020.11.035},
pmid = {33333018},
issn = {1097-4164},
abstract = {RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an &quot;asymmetric homodimer.&quot; Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.},
}
@article {pmid33332569,
year = {2020},
author = {Gabel, C and Li, Z and Zhang, H and Chang, L},
title = {Structural basis for inhibition of the type I-F CRISPR-Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa1199},
pmid = {33332569},
issn = {1362-4962},
abstract = {CRISPR-Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR-Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR-Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.},
}
@article {pmid33330425,
year = {2020},
author = {Meng, J and Qiu, Y and Shi, S},
title = {CRISPR/Cas9 Systems for the Development of Saccharomyces cerevisiae Cell Factories.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {594347},
pmid = {33330425},
issn = {2296-4185},
abstract = {Synthetic yeast cell factories provide a remarkable solution for the sustainable supply of a range of products, ranging from large-scale industrial chemicals to high-value pharmaceutical compounds. Synthetic biology is a field in which metabolic pathways are intensively studied and engineered. The clustered, regularly interspaced, short, palindromic repeat-associated (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has emerged as the state-of-the-art gene editing technique for synthetic biology. Recently, the use of different CRISPR/Cas9 systems has been extended to the field of yeast engineering for single-nucleotide resolution editing, multiple-gene editing, transcriptional regulation, and genome-scale modifications. Such advancing systems have led to accelerated microbial engineering involving less labor and time and also enhanced the understanding of cellular genetics and physiology. This review provides a brief overview of the latest research progress and the use of CRISPR/Cas9 systems in genetic manipulation, with a focus on the applications of Saccharomyces cerevisiae cell factory engineering.},
}
@article {pmid33329659,
year = {2020},
author = {An, Y and Geng, Y and Yao, J and Fu, C and Lu, M and Wang, C and Du, J},
title = {Efficient Genome Editing in Populus Using CRISPR/Cas12a.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {593938},
pmid = {33329659},
issn = {1664-462X},
abstract = {The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.},
}
@article {pmid33329634,
year = {2020},
author = {Modrzejewski, D and Hartung, F and Lehnert, H and Sprink, T and Kohl, C and Keilwagen, J and Wilhelm, R},
title = {Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {574959},
pmid = {33329634},
issn = {1664-462X},
abstract = {CRISPR/Cas enables a targeted modification of DNA sequences. Despite their ease and efficient use, one limitation is the potential occurrence of associated off-target effects. This systematic review aims to answer the following research question: Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas in plants? Literature published until March 2019 was considered for this review. Articles were screened for relevance based on pre-defined inclusion criteria. Relevant studies were subject to critical appraisal. All studies included in the systematic review were synthesized in a narrative report, but studies rated as high and medium/high validity were reported separately from studies rated as low and medium/low or unclear validity. In addition, we ran a binary logistic regression analysis to verify five factors that may affect the occurrence of off-target effects: (1) Number of mismatches (2) Position of mismatches (3) GC-content of the targeting sequence (4) Altered nuclease variants (5) Delivery methods. In total, 180 relevant articles were included in this review containing 468 studies therein. Seventy nine percentage of these studies were rated as having high or medium/high validity. Within these studies, 6,416 potential off-target sequences were assessed for the occurrence of off-target effects. Results clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence steeply decreases the likelihood of off-target effects. The observed rate of off-target effects decreased from 59% when there is one mismatch between the on-target and off-target sequences toward 0% when four or more mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decreased the occurrence of off-target effects. There is no evidence that the GC-content significantly affects off-target effects. The database regarding the impact of the nuclease variant and the delivery method is very poor as the majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Hence, a general significant impact of these two factors on the occurrence of off-target effects cannot be proved. This identified evidence gap needs to be filled by systematic studies exploring these individual factors in sufficient numbers.},
}
@article {pmid33328654,
year = {2021},
author = {Shivram, H and Cress, BF and Knott, GJ and Doudna, JA},
title = {Controlling and enhancing CRISPR systems.},
journal = {Nature chemical biology},
volume = {17},
number = {1},
pages = {10-19},
pmid = {33328654},
issn = {1552-4469},
support = {HR0011- 17-2-0043//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; HR0011- 17-2-0043//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; F32 GM131654/GM/NIGMS NIH HHS/United States ; 1175568//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
abstract = {Many bacterial and archaeal organisms use clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) systems to defend themselves from mobile genetic elements. These CRISPR-Cas systems are classified into six types based on their composition and mechanism. CRISPR-Cas enzymes are widely used for genome editing and offer immense therapeutic opportunity to treat genetic diseases. To realize their full potential, it is important to control the timing, duration, efficiency and specificity of CRISPR-Cas enzyme activities. In this Review we discuss the mechanisms of natural CRISPR-Cas regulatory biomolecules and engineering strategies that enhance or inhibit CRISPR-Cas immunity by altering enzyme function. We also discuss the potential applications of these CRISPR regulators and highlight unanswered questions about their evolution and purpose in nature.},
}
@article {pmid33323021,
year = {2020},
author = {Li, F and Zhou, C and Tu, T and Liu, Y and Lv, X and Wang, B and Song, Z and Zhao, Q and Liu, C and Gu, F and Zhao, J},
title = {Rational selection of CRISPR-Cas triggering homologous recombination directed repair in human cells.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2020.247},
pmid = {33323021},
issn = {1557-7422},
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated 9) nucleases have been widely applied for genome engineering. Cas9 (Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9)) and Cpf1 (i.e., Francisella novicida U112 Cpf1 (FnCpf1), also named FnCas12a) were harnessed to perform gene editing in human cells. Precise genetic modification by homologous recombination directed repair (HDR) is an attractive approach for in situ gene correction. However, so far, the comparative efficiencies of HDR mediated by different CRISPR orthologs remain unknown. To address this question here we developed a reporter system to investigate HDR efficiencies triggered by various CRISPR orthologs. We found that SpCas9 and SaCas9, the most two commonly used Cas9 enzymes, possessed a similar ability to induce HDR. Interestingly, with the increasing amount of coding plasmids or additional nuclear localization sequences (NLS), FnCpf1 could improve the HDR efficiency. Collectively, our study provides insights for the rational selection of appropriate tools for human genome manipulation.},
}
@article {pmid33321968,
year = {2020},
author = {Alayande, KA and Aiyegoro, OA and Ateba, CN},
title = {Distribution of Important Probiotic Genes and Identification of the Biogenic Amines Produced by Lactobacillus acidophilus PNW3.},
journal = {Foods (Basel, Switzerland)},
volume = {9},
number = {12},
pages = {},
pmid = {33321968},
issn = {2304-8158},
abstract = {The genome of Lactobacillus acidophilus PNW3 was assessed for probiotic and safety potentials. The genome was completely sequenced, assembled using SPAdes, and thereafter annotated with NCBI prokaryotic genome annotation pipeline (PGAP) and rapid annotation using subsystem technology (RAST). Further downstream assessment was determined using appropriate bioinformatics tools. The production of biogenic amines was confirmed through HPLC analysis and the evolutionary trend of the strain was determined through the Codon Tree pipeline. The strain was predicted as a non-human pathogen. A plethora of encoding genes for lactic acids and bioactive peptides production, adhesion molecules, resistance to the harsh gut environmental conditions, and improvement of the host metabolism, which are putative for important probiotic functionalities, were located at different loci within the genome. A bacteriocin predicted to be helveticin J was identified as one of the secondary metabolites. The maximum zone of inhibition exhibited by the crude bacteriocin against STEC E. coli O177 was 21.7 ± 0.58 mm and 24.3 ± 1.15 mm after partial purification (250 µg/mL). Three coding sequences were identified for insertion sequences and one for the CRISPR-Cas fragment. The protein-encoding sequence for Ornithine decarboxylase was found within the genome. L. acidophilus PNW3 presents important features categorizing it as a viable and safe probiotic candidate, though further safety investigations are necessary. The application of probiotics in livestock-keeping would ensure improved public health and food security.},
}
@article {pmid33321741,
year = {2020},
author = {Wu, J and Mukama, O and Wu, W and Li, Z and Habimana, JD and Zhang, Y and Zeng, R and Nie, C and Zeng, L},
title = {A CRISPR/Cas12a Based Universal Lateral Flow Biosensor for the Sensitive and Specific Detection of African Swine-Fever Viruses in Whole Blood.},
journal = {Biosensors},
volume = {10},
number = {12},
pages = {},
pmid = {33321741},
issn = {2079-6374},
support = {31671933//National Natural Science Foundation of China/ ; 911148427033//Guangdong Fund Committee for Basic and Applied Basic Research/ ; },
abstract = {Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful; however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR-Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 × 10-15 M within 2 h, and unambiguously identified ASFV from swine blood. This system uses less time for PCR pre-amplification and requires cheaper devices; thus, it can be applied to virus monitoring and food samples detection.},
}
@article {pmid33320883,
year = {2020},
author = {Xiong, D and Dai, W and Gong, J and Li, G and Liu, N and Wu, W and Pan, J and Chen, C and Jiao, Y and Deng, H and Ye, J and Zhang, X and Huang, H and Li, Q and Xue, L and Zhang, X and Tang, G},
title = {Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.},
journal = {PLoS biology},
volume = {18},
number = {12},
pages = {e3000978},
doi = {10.1371/journal.pbio.3000978},
pmid = {33320883},
issn = {1545-7885},
mesh = {Base Sequence ; COVID-19 Testing/*methods ; CRISPR-Cas Systems/*genetics ; Humans ; Reproducibility of Results ; SARS-CoV-2/*genetics/*isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.},
}
@article {pmid33317042,
year = {2020},
author = {Lucere, KM and O'Malley, MMR and Diermeier, SD},
title = {Functional Screening Techniques to Identify Long Non-Coding RNAs as Therapeutic Targets in Cancer.},
journal = {Cancers},
volume = {12},
number = {12},
pages = {},
pmid = {33317042},
issn = {2072-6694},
support = {RDF-UOO1802//Royal Society Te Apārangi/ ; },
abstract = {Recent technological advancements such as CRISPR/Cas-based systems enable multiplexed, high-throughput screening for new therapeutic targets in cancer. While numerous functional screens have been performed on protein-coding genes to date, long non-coding RNAs (lncRNAs) represent an emerging class of potential oncogenes and tumor suppressors, with only a handful of large-scale screens performed thus far. Here, we review in detail currently available screening approaches to identify new lncRNA drivers of tumorigenesis and tumor progression. We discuss the various approaches of genomic and transcriptional targeting using CRISPR/Cas9, as well as methods to post-transcriptionally target lncRNAs via RNA interference (RNAi), antisense oligonucleotides (ASOs) and CRISPR/Cas13. We discuss potential advantages, caveats and future applications of each method to provide an overview and guide on investigating lncRNAs as new therapeutic targets in cancer.},
}
@article {pmid33315879,
year = {2020},
author = {Petrozziello, T and Dios, AM and Mueller, KA and Vaine, CA and Hendriks, WT and Glajch, KE and Mills, AN and Mangkalaphiban, K and Penney, EB and Ito, N and Fernandez-Cerado, C and Legarda, GPA and Velasco-Andrada, MS and Acuña, PJ and Ang, MA and Muñoz, EL and Diesta, CCE and Macalintal-Canlas, R and Acuña, G and Sharma, N and Ozelius, LJ and Bragg, DC and Sadri-Vakili, G},
title = {SVA insertion in X-linked Dystonia Parkinsonism alters histone H3 acetylation associated with TAF1 gene.},
journal = {PloS one},
volume = {15},
number = {12},
pages = {e0243655},
pmid = {33315879},
issn = {1932-6203},
abstract = {X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.},
}
@article {pmid33309323,
year = {2020},
author = {Le, TK and Paris, C and Khan, KS and Robson, F and Ng, WL and Rocchi, P},
title = {Nucleic Acid-Based Technologies Targeting Coronaviruses.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
pmid = {33309323},
issn = {0968-0004},
abstract = {The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently creating a global health emergency. This crisis is driving a worldwide effort to develop effective vaccines, prophylactics, and therapeutics. Nucleic acid (NA)-based treatments hold great potential to combat outbreaks of coronaviruses (CoVs) due to their rapid development, high target specificity, and the capacity to increase druggability. Here, we review key anti-CoV NA-based technologies, including antisense oligonucleotides (ASOs), siRNAs, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas), and mRNA vaccines, and discuss improved delivery methods and combination therapies with other antiviral drugs.},
}
@article {pmid33307189,
year = {2020},
author = {Nishida, K and Kondo, A},
title = {CRISPR-derived genome editing technologies for metabolic engineering.},
journal = {Metabolic engineering},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymben.2020.12.002},
pmid = {33307189},
issn = {1096-7184},
abstract = {In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.},
}
@article {pmid33306075,
year = {2020},
author = {Sha, Y and Huang, R and Huang, M and Yue, H and Shan, Y and Hu, J and Xing, D},
title = {Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d0cc06412b},
pmid = {33306075},
issn = {1364-548X},
abstract = {Existing CRISPR/cas-based biosensors usually improve sensitivity by target amplification, which is time-consuming and susceptible to impurities in complex biofluid. Herein, this is the first time a cascade CRISPR/cas (casCRISPR) system has been developed, which can provide a detection limit of 1.33 fM (∼1000 times lower than direct Cas13a-based miRNA detection) and single-base resolution for miR-17 detection without resorting to target amplification. casCRISPR can also be applied to detect miRNA in complicated cell extracts and serum samples. Overall, casCRISPR will provide a heuristic idea for CRISPR/cas based biosensing, and could be a promising tool for miRNA diagnostics.},
}
@article {pmid33304462,
year = {2020},
author = {Antao, AM and Karapurkar, JK and Lee, DR and Kim, KS and Ramakrishna, S},
title = {Disease modeling and stem cell immunoengineering in regenerative medicine using CRISPR/Cas9 systems.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {3649-3665},
pmid = {33304462},
issn = {2001-0370},
abstract = {CRISPR/Cas systems are popular genome editing tools that belong to a class of programmable nucleases and have enabled tremendous progress in the field of regenerative medicine. We here outline the structural and molecular frameworks of the well-characterized type II CRISPR system and several computational tools intended to facilitate experimental designs. The use of CRISPR tools to generate disease models has advanced research into the molecular aspects of disease conditions, including unraveling the molecular basis of immune rejection. Advances in regenerative medicine have been hindered by major histocompatibility complex-human leukocyte antigen (HLA) genes, which pose a major barrier to cell- or tissue-based transplantation. Based on progress in CRISPR, including in recent clinical trials, we hypothesize that the generation of universal donor immune-engineered stem cells is now a realistic approach to tackling a multitude of disease conditions.},
}
@article {pmid33300275,
year = {2020},
author = {Derry, WB},
title = {CRISPR: development of a technology and its applications.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.15621},
pmid = {33300275},
issn = {1742-4658},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) is a prokaryotic immune surveillance system that is used by bacteria to recognize genetic material of infectious organisms, such as phage viruses. Using CRISPR-associated (Cas) proteins, this system cleaves foreign nucleic acid into fragments, thus defending the bacterium against the attacker. The 2020 Nobel Prize in Chemistry was awarded to CRISPR-Cas pioneers Emmanuelle Charpentier and Jennifer Doudna, who developed the CRISPR-Cas system to precisely edit genomic DNA. This technology has exploded at a breathtaking pace and is now used by almost every molecular biology laboratory around the world in a myriad of organisms. In this Virtual Issue, the FEBS Journal features articles reviewing the development of CRISPR/Cas9 technology and its applications to understand the functions of proteins in vivo.},
}
@article {pmid33299151,
year = {2020},
author = {Schwartz, C and Lenderts, B and Feigenbutz, L and Barone, P and Llaca, V and Fengler, K and Svitashev, S},
title = {CRISPR-Cas9-mediated 75.5-Mb inversion in maize.},
journal = {Nature plants},
volume = {6},
number = {12},
pages = {1427-1431},
pmid = {33299151},
issn = {2055-0278},
abstract = {CRISPR-Cas is a powerful double-strand-break technology with wide-ranging applications from gene discovery to commercial product development. Thus far, this tool has been almost exclusively used for gene knockouts and deletions, with a few examples of gene edits and targeted gene insertions. Here, we demonstrate the application of CRISPR-Cas9 technology to mediate targeted 75.5-Mb pericentric inversion in chromosome 2 in one of the elite maize inbred lines from Corteva Agriscience. This inversion unlocks a large chromosomal region containing substantial genetic variance for recombination, thus providing opportunities for the development of new maize varieties with improved phenotypes.},
}
@article {pmid33298251,
year = {2020},
author = {Kim, S and Hupperetz, C and Lim, S and Kim, CH},
title = {Genome editing of immune cells using CRISPR/Cas9.},
journal = {BMB reports},
volume = {},
number = {},
pages = {},
pmid = {33298251},
issn = {1976-670X},
abstract = {The ability to read, write, and edit genomic information in living organisms can have a profound impact on research, health, economic, and environmental issues. The CRISPR/Cas system, recently discovered as an adaptive immune system in prokaryotes, has revolutionized the ease and throughput of genome editing in mammalian cells and has proved itself indispensable to the engineering of immune cells and identification of novel immune mechanisms. In this review, we summarize the CRISPR/Cas9 system and the history of its discovery and optimization. We then focus on engineering T cells and other types of immune cells, with emphasis on therapeutic applications. Last, we describe the different modifications of Cas9 and their recent applications in the genome-wide screening of immune cells.},
}
@article {pmid33295064,
year = {2020},
author = {Xiong, E and Jiang, L and Tian, T and Hu, M and Yue, H and Huang, M and Lin, W and Jiang, Y and Zhu, D and Zhou, X},
title = {Simultaneous dual-gene diagnosis of SARS-CoV-2 based on CRISPR/Cas9-mediated lateral flow assay.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {},
doi = {10.1002/anie.202014506},
pmid = {33295064},
issn = {1521-3773},
abstract = {Accurate and large-scale screening of infected individuals has proven to be an effective means to control the spread of COVID-19. Currently, many assays have been developed to meet the huge testing requirements and the availability of diverse detection settings. However, few methods emphasize the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Herein, we developed a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (ORF1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab clinical samples showed 100% negative predictive agreement (NPA) and 97.14% positive predictive agreement (PPA). Expectantly, this developed platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.},
}
@article {pmid33294131,
year = {2020},
author = {Shibata, T and Iwasaki, W and Hirota, K},
title = {The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {3350-3360},
pmid = {33294131},
issn = {2001-0370},
abstract = {Double-stranded (ds)DNA, not dsRNA, has an ability to form a homologous complex with single-stranded (ss)DNA or ssRNA of homologous sequence. D-loops and homologous triplexes are homologous complexes formed with ssDNA by RecA/Rad51-family homologous-pairing proteins, and are a key intermediate of homologous (genetic/DNA) recombination. R-loop formation independent of transcription (R-loop formation in trans) was recently found to play roles in gene regulation and development of mammals and plants. In addition, the crRNA-Cas effector complex in CRISPR-Cas systems also relies on R-loop formation to recognize specific target. In homologous complex formation, ssDNA/ssRNA finds a homologous sequence in dsDNA by Watson-Crick base-pairing. crRNA-Cas effector complexes appear to actively melt dsDNA to make its bases available for annealing to crRNA. On the other hand, in D-loop formation and homologous-triplex formation, it is likely that dsDNA recognizes the homologous sequence before the melting of its double helix by using its intrinsic molecular function depending on CH2 at the 2'-position of the deoxyribose, and that the major role of RecA is the extension of ssDNA and the holding dsDNA at a position suitable for homology search. This intrinsic dsDNA function would also play a role in R-loop formation. The dependency of homologous-complex formation on 2'-CH2 of the deoxyribose would explain the absence of homologous complex formation by dsRNA, and dsDNA as sole genome molecule in all cellular organisms.},
}
@article {pmid33293368,
year = {2020},
author = {Nakato, M and Shiranaga, N and Tomioka, M and Watanabe, H and Kurisu, J and Kengaku, M and Komura, N and Ando, H and Kimura, Y and Kioka, N and Ueda, K},
title = {ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA120.015997},
pmid = {33293368},
issn = {1083-351X},
abstract = {ATP-binding cassette subfamily A member 13 (ABCA13) is predicted to be the largest ABC protein, consisting of 5,058 amino acids and a long N-terminal region. Mutations in the ABCA13 gene were reported to increase the susceptibility to schizophrenia, bipolar disorder and major depression. However, little is known about the molecular functions of ABCA13 or how they associate with psychiatric disorders. Here, we examined the biochemical activity of ABCA13 using HEK293 cells transfected with mouse ABCA13. The expression of ABCA13 induced the internalization of cholesterol and gangliosides from the plasma membrane to intracellular vesicles. Cholesterol internalization by ABCA13 required the long N-terminal region and ATP hydrolysis. To examine the physiological roles of ABCA13, we generated Abca13 KO mice using CRISPR/Cas and found that these mice exhibited deficits of prepulse inhibition. Vesicular cholesterol accumulation and synaptic vesicle endocytosis were impaired in primary cultures of Abca13 KO cortical neurons. Furthermore, mutations in ABCA13 gene associated with psychiatric disorders disrupted the protein's subcellular localization and impaired cholesterol trafficking. These findings suggest that ABCA13 accelerates cholesterol internalization by endocytic retrograde transport in neurons and that loss-of-this function is associated with the pathophysiology of psychiatric disorders.},
}
@article {pmid33292223,
year = {2020},
author = {Miri, SM and Tafsiri, E and Cho, WCS and Ghaemi, A},
title = {Correction to: CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy.},
journal = {Cancer cell international},
volume = {20},
number = {1},
pages = {521},
doi = {10.1186/s12935-020-01609-w},
pmid = {33292223},
issn = {1475-2867},
abstract = {An amendment to this paper has been published and can be accessed via the original article.},
}
@article {pmid33291011,
year = {2020},
author = {Ovchinnikov, DA},
title = {Genetically-modified cell lines: categorisation and considerations for characterisation.},
journal = {Stem cell research},
volume = {49},
number = {},
pages = {102103},
doi = {10.1016/j.scr.2020.102103},
pmid = {33291011},
issn = {1876-7753},
abstract = {Stem Cell Research is pleased to introduce into its publication portfolio a new article type: a template-driven short report on the generation of a novel Genetically Modified Cell Line. This resource type is typically derived from human pluripotent stem cell lines via the introduction of nucleases and/or foreign genetic material leading to stable genomic alterations, maintained in a single cell-derived clonal cell line. Interest in, and demand for, genetically modified cell lines has grown exponentially in the last few years. This overview provides a brief introduction to this incredibly versatile lab resource and marks the beginning of a new and exciting addition to the publication portfolio of Stem Cell Research. A dramatic increase in the accessibility of the human genome in the last decade has given a long-anticipated boost to advanced biomedical studies in human in vitro systems. Pluripotent stem cells represent a particularly attractive gateway into this line of experimentation due to their unique suitability for the isolation of clonal genetically modified cell lines (GMCLs), and the ability to be differentiated into essentially any cell type upon the lines' virtually limitless expansion.},
}
@article {pmid33290744,
year = {2020},
author = {Songailiene, I and Juozapaitis, J and Tamulaitiene, G and Ruksenaite, A and Šulčius, S and Sasnauskas, G and Venclovas, Č and Siksnys, V},
title = {HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation.},
journal = {Molecular cell},
volume = {80},
number = {6},
pages = {955-970.e7},
doi = {10.1016/j.molcel.2020.11.034},
pmid = {33290744},
issn = {1097-4164},
abstract = {Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.},
}
@article {pmid33290505,
year = {2020},
author = {Mitrofanov, A and Alkhnbashi, OS and Shmakov, SA and Makarova, KS and Koonin, EV and Backofen, R},
title = {CRISPRidentify: identification of CRISPR arrays using machine learning approach.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa1158},
pmid = {33290505},
issn = {1362-4962},
abstract = {CRISPR-Cas are adaptive immune systems that degrade foreign genetic elements in archaea and bacteria. In carrying out their immune functions, CRISPR-Cas systems heavily rely on RNA components. These CRISPR (cr) RNAs are repeat-spacer units that are produced by processing of pre-crRNA, the transcript of CRISPR arrays, and guide Cas protein(s) to the cognate invading nucleic acids, enabling their destruction. Several bioinformatics tools have been developed to detect CRISPR arrays based solely on DNA sequences, but all these tools employ the same strategy of looking for repetitive patterns, which might correspond to CRISPR array repeats. The identified patterns are evaluated using a fixed, built-in scoring function, and arrays exceeding a cut-off value are reported. Here, we instead introduce a data-driven approach that uses machine learning to detect and differentiate true CRISPR arrays from false ones based on several features. Our CRISPR detection tool, CRISPRidentify, performs three steps: detection, feature extraction and classification based on manually curated sets of positive and negative examples of CRISPR arrays. The identified CRISPR arrays are then reported to the user accompanied by detailed annotation. We demonstrate that our approach identifies not only previously detected CRISPR arrays, but also CRISPR array candidates not detected by other tools. Compared to other methods, our tool has a drastically reduced false positive rate. In contrast to the existing tools, our approach not only provides the user with the basic statistics on the identified CRISPR arrays but also produces a certainty score as a practical measure of the likelihood that a given genomic region is a CRISPR array.},
}
@article {pmid33289251,
year = {2020},
author = {Schneider, A},
title = {A short history of guide RNAs: The intricate path that led to the discovery of a basic biological concept.},
journal = {EMBO reports},
volume = {},
number = {},
pages = {e51918},
pmid = {33289251},
issn = {1469-3178},
support = {NCCR RNA &amp; Disease//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (SNF)/ ; 175563//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (SNF)/ ; },
abstract = {This year's Nobel prize for the CRISPR/Cas system is an illustrative example of how scientific breakthroughs rests on preceding work: the discovery of guide RNAs in the 1990s.},
}
@article {pmid33287207,
year = {2020},
author = {Shaidullina, E and Shelenkov, A and Yanushevich, Y and Mikhaylova, Y and Shagin, D and Alexandrova, I and Ershova, O and Akimkin, V and Kozlov, R and Edelstein, M},
title = {Antimicrobial Resistance and Genomic Characterization of OXA-48- and CTX-M-15-Co-Producing Hypervirulent Klebsiella pneumoniae ST23 Recovered from Nosocomial Outbreak.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {9},
number = {12},
pages = {},
pmid = {33287207},
issn = {2079-6382},
support = {075-15-2019-1666//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Multidrug resistance (MDR) and hypervirulence (hv) have been long considered distinct evolutionary traits for Klebsiella pneumoniae (Kp), a versatile human pathogen. The recent emergence of Kp strains combining these traits poses a serious global threat. In this article, we describe the phenotypic and genomic characteristics of an MDR hvKp isolate, MAR14-456, representative of a nosocomial outbreak in Moscow, Russia, that was recovered from a postoperative wound in a patient who later developed multiple abscesses, fatal sepsis, and septic shock. Broth microdilution testing revealed decreased susceptibility of MAR14-456 to carbapenems (MICs 0.5-2 mg/L) and a high-level resistance to most β-lactams, β-lactam-β-lactamase-inhibitor combinations, and non-β-lactam antibiotics, except ceftazidime-avibactam, amikacin, tigecycline, and colistin. Whole-genome sequencing using Illumina MiSeq and ONT MinION systems allowed to identify and completely assemble two conjugative resistance plasmids, a typical 'European' epidemic IncL/M plasmid that carries the gene of OXA-48 carbapenemase, and an IncFIIK plasmid that carries the gene of CTX-M-15 ESBL and other resistance genes. MLST profile, capsular, lipopolysaccharide, virulence genes encoded on chromosome and IncHI1B/FIB plasmid, and the presence of apparently functional type I-E* CRISPR-Cas system were all characteristic of hvKp ST23, serotype K1-O1v2. Phylogenetic analysis showed the closest relatedness of MAR14-456 to ST23 isolates from China. This report highlights the threat of multiple resistance acquisition by hvKp strain and its spread as a nosocomial pathogen.},
}
@article {pmid33285226,
year = {2020},
author = {Dey, A},
title = {CRISPR/Cas genome editing to optimize pharmacologically active plant natural products.},
journal = {Pharmacological research},
volume = {},
number = {},
pages = {105359},
doi = {10.1016/j.phrs.2020.105359},
pmid = {33285226},
issn = {1096-1186},
abstract = {Since time immemorial, human use medicinal plants as sources of food, therapy and industrial purpose. Classical biotechnology and recent next-generation sequencing (NGS) techniques have been successfully used to optimize plant-derived natural-products of biomedical significance. Earlier, protein based editing tools viz. zinc-finger nucleases (ZFNs) and transcription activator-like endonucleases (TALENs) have been popularized for transcriptional level genome manipulation. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is an efficient, robust and selective site-directed mutagenesis strategy for RNA-guided genome-editing. CRISPR/Cas9 genome-editing tool employs designed guide-RNAs that identifies a 3 base-pair protospacer adjacent motif (PAM) sequence occurring downstream of the target-DNA. The present review comprehensively complies the recent literature (2010-2020) retrieved from scientific-databases on the application of CRISPR/Cas9-editing-tools as potent genome-editing strategies in medicinal-plants discussing the recent developments, challenges and future-perspectives with notes on broader applicability of the technique in plants and lower-organisms. In plants, CRISPR/Cas-editing has been implemented successfully in relation to crop-yield and stress-tolerance. However, very few medicinal plants have been edited using CRISPR/Cas genome tool owing to the lack of whole-genome and mRNA-sequences and shortfall of suitable transformation and regeneration strategies. However, recently a number of plant secondary metabolic-pathways (viz. alkaloid, terpenoid, flavonoids, phenolic, saponin etc.) have been engineered employing CRISPR/Cas-editing via knock-out, knock-in, point-mutation, fine-tuning of gene-expression and targeted-mutagenesis. This genome-editing tool further extends its applicability incorporating the tools of synthetic- and systems-biology, functional-genomics and NGS to produce genetically-engineered medicinal-crops with advanced-traits facilitating the production of pharmaceuticals and nutraceuticals.},
}
@article {pmid33283798,
year = {2020},
author = {Bukasov, R and Dossym, D and Filchakova, O},
title = {Detection of RNA viruses from influenza and HIV to Ebola and SARS-CoV-2: a review.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d0ay01886d},
pmid = {33283798},
issn = {1759-9679},
abstract = {RNA-based viruses likely make up the highest pandemic threat among all known pathogens in about the last 100 years, since the Spanish Flu of 1918 with 50 M deaths up to COVID-19. Nowadays, an efficient and affordable testing strategy for such viruses have become the paramount target for the fields of virology and bioanalytical chemistry. The detection of the viruses (influenza, hepatitis, HIV, Zika, SARS, Ebola, SARS-CoV-2, etc.) and human antibodies to these viruses is described and tabulated in terms of the reported methods of detection, time to results, accuracy and specificity, if they are reported. The review is focused, but not limited to publications in the last decade. Finally, the limits of detection for each representative publication are tabulated by detection methods and discussed. These methods include PCR, lateral flow immunoassays, LAMP-based methods, ELISA, electrochemical methods (e.g., amperometry, voltammetry), fluorescence spectroscopy, AFM, SPR and SERS spectroscopy, silver staining and CRISPR-Cas based methods, bio-barcode detection, and resonance light scattering. The review is likely to be interesting for various scientists, and particularly helpful with information for establishing interdisciplinary research.},
}
@article {pmid33283789,
year = {2020},
author = {Louradour, I and Ghosh, K and Inbar, E and Sacks, DL and Aluvihare, C and Harrell, RA},
title = {Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {165},
pages = {},
doi = {10.3791/61924},
pmid = {33283789},
issn = {1940-087X},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Embryo, Nonmammalian/*metabolism ; Female ; Male ; Mice ; *Microinjections ; Microtechnology ; Mutagenesis/*genetics ; Mutation/genetics ; Needles ; Phlebotomus/*embryology/genetics/immunology/parasitology ; },
abstract = {Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.},
}
@article {pmid33282821,
year = {2020},
author = {Minarini, LADR and de Andrade, LN and De Gregorio, E and Grosso, F and Naas, T and Zarrilli, R and Camargo, ILBC},
title = {Editorial: Antimicrobial Resistance as a Global Public Health Problem: How Can We Address It?.},
journal = {Frontiers in public health},
volume = {8},
number = {},
pages = {612844},
pmid = {33282821},
issn = {2296-2565},
}
@article {pmid33280580,
year = {2020},
author = {Shemyakin, IG and Firstova, VV and Fursova, NK and Abaev, IV and Filippovich, SY and Ignatov, SG and Dyatlov, IA},
title = {Next-Generation Antibiotics, Bacteriophage Endolysins, and Nanomaterials for Combating Pathogens.},
journal = {Biochemistry. Biokhimiia},
volume = {85},
number = {11},
pages = {1374-1388},
doi = {10.1134/S0006297920110085},
pmid = {33280580},
issn = {1608-3040},
abstract = {This review presents various strategies to fight causative agents of infectious diseases. Species-specific programmable RNA-containing antibiotics open up new possibilities for creating next-generation of personalized drugs based on microbiome editing and can serve as a new tool for selective elimination of pathogenic bacterial species while keeping intact the rest of microbiota. Another promising approach in combating bacterial infections is genome editing using the CRISPR-Cas systems. Expanding knowledge on the molecular mechanisms of innate immunity has been actively used for developing new antimicrobials. However, obvious risks of using antibiotic adjuvants aimed at activation of the host immune system include development of the autoimmune response with subsequent organ damage. To avoid these risks, it is essential to elucidate action mechanisms of the specific ligands and signal molecules used as components of the hybrid antibiotics. Bacteriophage endolysins are also considered as effective antimicrobials against antibiotic-resistant bacteria, metabolically inactive persisters, and microbial biofilms. Despite significant advances in the design of implants with antibacterial properties, the problem of postoperative infections still remains. Different nanomodifications of the implant surface have been designed to reduce bacterial contamination. Here, we review bactericidal, fungicidal, and immunomodulating properties of compounds used for the implant surface nanomodifications, such as silver, boron nitride nanomaterials, nanofibers, and nanogalvanic materials.},
}
@article {pmid33277326,
year = {2020},
author = {Mukherjee, A},
title = {Mutagenomics: The Future of Genetic Screens.},
journal = {Plant physiology},
volume = {184},
number = {4},
pages = {1616-1617},
pmid = {33277326},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems ; *Genetic Testing ; Mutation ; },
}
@article {pmid33275853,
year = {2020},
author = {Russel, J and Pinilla-Redondo, R and Mayo-Muñoz, D and Shah, SA and Sørensen, SJ},
title = {CRISPRCasTyper: Automated Identification, Annotation, and Classification of CRISPR-Cas Loci.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {462-469},
doi = {10.1089/crispr.2020.0059},
pmid = {33275853},
issn = {2573-1602},
abstract = {Automated classification of CRISPR-Cas systems has been challenged by their dynamic nature and expanding classification. Here, we developed CRISPRCasTyper, an automated tool with improved capabilities for identifying and typing CRISPR arrays and cas loci based on the latest nomenclature (44 subtypes/variants). As a novel feature, CRISPRCasTyper uses a machine learning approach to subtype CRISPR arrays based on the sequences of the repeats, which allows the typing of orphan and distant arrays. CRISPRCasTyper provides a graphical output, where CRISPRs and cas operons are visualized as gene maps, thus aiding annotation of partial and novel systems through synteny. CRISPRCasTyper was benchmarked against a manually curated set of 31 subtypes with a median accuracy of 98.6% and used to explore CRISPR-Cas diversity across >3,000 metagenomes. Altogether, we present an up-to-date software for improved automated prediction of CRISPR-Cas loci. CRISPRCasTyper is available through conda and as a web server (cctyper.crispr.dk).},
}
@article {pmid33275752,
year = {2020},
author = {Basar, R and Daher, M and Rezvani, K},
title = {Next-generation cell therapies: the emerging role of CAR-NK cells.},
journal = {Hematology. American Society of Hematology. Education Program},
volume = {2020},
number = {1},
pages = {570-578},
pmid = {33275752},
issn = {1520-4383},
abstract = {T cells engineered with chimeric antigen receptors (CARs) have revolutionized the field of cell therapy and changed the paradigm of treatment for many patients with relapsed or refractory B-cell malignancies. Despite this progress, there are limitations to CAR-T cell therapy in both the autologous and allogeneic settings, including practical, logistical, and toxicity issues. Given these concerns, there is a rapidly growing interest in natural killer cells as alternative vehicles for CAR engineering, given their unique biological features and their established safety profile in the allogeneic setting. Other immune effector cells, such as invariant natural killer T cells, γδ T cells, and macrophages, are attracting interest as well and eventually may be added to the repertoire of engineered cell therapies against cancer. The pace of these developments will undoubtedly benefit from multiple innovative technologies, such as the CRISPR-Cas gene editing system, which offers great potential to enhance the natural ability of immune effector cells to eliminate refractory cancers.},
}
@article {pmid33274276,
year = {2020},
author = {Rauch, S and Jones, KA and Dickinson, BC},
title = {Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation.},
journal = {ACS central science},
volume = {6},
number = {11},
pages = {1987-1996},
pmid = {33274276},
issn = {2374-7943},
abstract = {All aspects of mRNA lifetime and function, including its stability, translation into protein, and trafficking through the cell, are tightly regulated through coordinated post-transcriptional modifications and interactions with a multitude of RNA effector proteins. Despite the increasing recognition of RNA regulation as a critical layer of mammalian gene expression control and its increasing excitement as a therapeutic target, tools to study and control RNA regulatory mechanisms with temporal precision in their endogenous environment are lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired RNA targeting system (CIRTS). The CIRTS biosensor platform is based on guide RNA (gRNA)-dependent RNA binding domains that interact with a target transcript using Watson-Crick-Franklin base pair interactions. Addition of a small molecule recruits an RNA effector to the target transcript, thereby eliciting a local effect on the transcript. In this work, we showcase that these CIRTS biosensors can trigger inducible RNA editing, degradation, or translation on target transcripts in a small molecule-dependent manner. We further go on to show that the CIRTS RNA base editor biosensor can induce RNA base editing in a small molecule-controllable manner in vivo. Collectively this work provides a new set of tools to probe the dynamics of RNA regulatory systems and control gene expression at the RNA level.},
}
@article {pmid33273523,
year = {2020},
author = {Kushwaha, SK and Bhavesh, NLS and Abdella, B and Lahiri, C and Marathe, SA},
title = {The phylogenomics of CRISPR-Cas system and revelation of its features in Salmonella.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {21156},
pmid = {33273523},
issn = {2045-2322},
abstract = {Salmonellae display intricate evolutionary patterns comprising over 2500 serovars having diverse pathogenic profiles. The acquisition and/or exchange of various virulence factors influences the evolutionary framework. To gain insights into evolution of Salmonella in association with the CRISPR-Cas genes we performed phylogenetic surveillance across strains of 22 Salmonella serovars. The strains differed in their CRISPR1-leader and cas operon features assorting into two main clades, CRISPR1-STY/cas-STY and CRISPR1-STM/cas-STM, comprising majorly typhoidal and non-typhoidal Salmonella serovars respectively. Serovars of these two clades displayed better relatedness, concerning CRISPR1-leader and cas operon, across genera than between themselves. This signifies the acquisition of CRISPR1/Cas region could be through a horizontal gene transfer event owing to the presence of mobile genetic elements flanking CRISPR1 array. Comparison of CRISPR and cas phenograms with that of multilocus sequence typing (MLST) suggests differential evolution of CRISPR/Cas system. As opposed to broad-host-range, the host-specific serovars harbor fewer spacers. Mapping of protospacer sources suggested a partial correlation of spacer content with habitat diversity of the serovars. Some serovars like serovar Enteritidis and Typhimurium that inhabit similar environment/infect similar hosts hardly shared their protospacer sources.},
}
@article {pmid33271061,
year = {2020},
author = {Petassi, MT and Hsieh, SC and Peters, JE},
title = {Guide RNA Categorization Enables Target Site Choice in Tn7-CRISPR-Cas Transposons.},
journal = {Cell},
volume = {183},
number = {7},
pages = {1757-1771.e18},
doi = {10.1016/j.cell.2020.11.005},
pmid = {33271061},
issn = {1097-4172},
abstract = {CRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways: one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Multiple mechanisms allow functionally distinct guide RNAs for transposition: a conventional system capable of acquiring guide RNAs to new plasmid and phage targets and a second providing long-term memory for access to chromosomal sites upon entry into a new host. Guide RNAs are privatized to be recognized only by the transposon-adapted system via sequence specialization, mismatch tolerance, and selective regulation to avoid toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals promising avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.},
}
@article {pmid33268788,
year = {2020},
author = {Coradini, ALV and Hull, CB and Ehrenreich, IM},
title = {Building genomes to understand biology.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6177},
pmid = {33268788},
issn = {2041-1723},
support = {R35 GM130381/GM/NIGMS NIH HHS/United States ; T32 GM118289/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Chimerism ; Chromosomes, Artificial/*chemistry/metabolism ; DNA/*genetics/metabolism ; Escherichia coli/genetics/metabolism ; Genetic Code ; *Genome ; Genomics/methods/*trends ; Humans ; Plasmids/chemistry/metabolism ; Saccharomyces cerevisiae/genetics/metabolism ; Synthetic Biology/methods/*trends ; },
abstract = {Genetic manipulation is one of the central strategies that biologists use to investigate the molecular underpinnings of life and its diversity. Thus, advances in genetic manipulation usually lead to a deeper understanding of biological systems. During the last decade, the construction of chromosomes, known as synthetic genomics, has emerged as a novel approach to genetic manipulation. By facilitating complex modifications to chromosome content and structure, synthetic genomics opens new opportunities for studying biology through genetic manipulation. Here, we discuss different classes of genetic manipulation that are enabled by synthetic genomics, as well as biological problems they each can help solve.},
}
@article {pmid33268787,
year = {2020},
author = {Lu, Z and Guo, JK and Wei, Y and Dou, DR and Zarnegar, B and Ma, Q and Li, R and Zhao, Y and Liu, F and Choudhry, H and Khavari, PA and Chang, HY},
title = {Structural modularity of the XIST ribonucleoprotein complex.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6163},
pmid = {33268787},
issn = {2041-1723},
support = {R01 HG004361/HG/NHGRI NIH HHS/United States ; RM1 HG007735/HG/NHGRI NIH HHS/United States ; R00 HG009662/HG/NHGRI NIH HHS/United States ; P30 CA014089/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenine/*analogs &amp; derivatives/metabolism ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Conserved Sequence ; Cross-Linking Reagents ; Female ; Ficusin/chemistry ; Formaldehyde/chemistry ; Gene Knock-In Techniques ; Humans ; K562 Cells ; Male ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Nucleic Acid Conformation ; Pregnancy ; RNA, Long Noncoding/*chemistry/genetics/metabolism ; Ribonucleoproteins/*chemistry/genetics/metabolism ; Sequence Analysis, RNA ; },
abstract = {Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. XIST controls the inactivation of an entire X chromosome in female placental mammals. Here we develop and integrate several orthogonal structure-interaction methods to demonstrate that XIST RNA-protein complex folds into an evolutionarily conserved modular architecture. Chimeric RNAs and clustered protein binding in fRIP and eCLIP experiments align with long-range RNA secondary structure, revealing discrete XIST domains that interact with distinct sets of effector proteins. CRISPR-Cas9-mediated permutation of the Xist A-repeat location shows that A-repeat serves as a nucleation center for multiple Xist-associated proteins and m6A modification. Thus modular architecture plays an essential role, in addition to sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and suggests a general strategy for mechanistic studies of large ribonucleoprotein assemblies.},
}
@article {pmid33260774,
year = {2020},
author = {Papagiannitsis, CC and Verra, A and Galani, V and Xitsas, S and Bitar, I and Hrabak, J and Petinaki, E},
title = {Unravelling the Features of Success of VIM-Producing ST111 and ST235 Pseudomonas aeruginosa in a Greek Hospital.},
journal = {Microorganisms},
volume = {8},
number = {12},
pages = {},
pmid = {33260774},
issn = {2076-2607},
support = {CZ.02.1.01/0.0/0.0/16_019/0000787//Ministerstvo Školství, Mládeže a Tělovýchovy/ ; },
abstract = {The objective of this study was to analyze the characteristics that contribute to the successful dissemination of VIM-producing Pseudomonas aeruginosa (P. aeruginosa), belonging to ST111 and ST235, in a Greek hospital. A total of 120 non-repetitive P. aeruginosa, which had meropenem minimal inhibitory concentrations (MICs) greater than 2 mg/L, were studied. VIM-encoding genes were amplified and sequenced within their integrons. Isolates were typed by multilocus sequence typing (MLST). Six VIM-producers, representative of different integron structures and sequence types (STs), were completely sequenced using Illumina platform. Sixty-one P. aeruginosa were confirmed to produce VIM-type carbapenemases. ST111 dominated (n = 34) among VIM-producers, while 15 VIM-producers belonged to ST235. The blaVIM-like genes were located in three integron types, including In59, In595 and In1760, which were integrated into P. aeruginosa chromosomes. Whole genome sequencing (WGS) data demonstrated that ST111 and ST235 MBL producers carried several resistance and virulence genes. Additionally, the presence of type I-C and type I-E clustered regularly interspaced short palindromic repeats (CRISPR)/Cas locus was observed in ST235 and ST395 isolates, respectively. In conclusion, our findings confirmed the clonal spread of ST111 P. aeruginosa, carrying the VIM-2-encoding integron In59, in the University Hospital of Larissa (UHL). In addition, they highlighted the important role of high-risk clones, ST111 and ST235, in the successful dissemination and establishment into hospital settings of clinically important pathogens carrying resistance determinants.},
}
@article {pmid33260366,
year = {2020},
author = {Ungefroren, H and Otterbein, H and Wellner, UF and Keck, T and Lehnert, H and Marquardt, JU},
title = {RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFβ1 in the Suppression of Cell Motility.},
journal = {Cancers},
volume = {12},
number = {12},
pages = {},
pmid = {33260366},
issn = {2072-6694},
abstract = {Autocrine transforming growth factor (TGF)β has been implicated in epithelial-mesenchymal transition (EMT) and invasion of several cancers including pancreatic ductal adenocarcinoma (PDAC) as well as triple-negative breast cancer (TNBC). However, the precise mechanism and the upstream inducers or downstream effectors of endogenous TGFB1 remain poorly characterized. In both cancer types, the small GTPase RAC1B inhibits cell motility induced by recombinant human TGFβ1 via downregulation of the TGFβ type I receptor, ALK5, but whether RAC1B also impacts autocrine TGFβ signaling has not yet been studied. Intriguingly, RNA interference-mediated knockdown (RNAi-KD) or CRISPR/Cas-mediated knockout of RAC1B in TGFβ1-secreting PDAC-derived Panc1 cells resulted in a dramatic decrease in secreted bioactive TGFβ1 in the culture supernatants and TGFB1 mRNA expression, while the reverse was true for TNBC-derived MDA-MB-231 cells ectopically expressing RAC1B. Surprisingly, the antibody-mediated neutralization of secreted bioactive TGFβ or RNAi-KD of the endogenous TGFB1 gene, was associated with increased rather than decreased migratory activities of Panc1 and MDA-MB-231 cells, upregulation of the promigratory genes SNAI1, SNAI2 and RAC1, and downregulation of the invasion suppressor genes CDH1 (encoding E-cadherin) and SMAD3. Intriguingly, ectopic re-expression of SMAD3 was able to rescue Panc1 and MDA-MB-231 cells from the TGFB1 KD-induced rise in migratory activity. Together, these data suggest that RAC1B favors synthesis and secretion of autocrine TGFβ1 which in a SMAD3-dependent manner blocks EMT-associated gene expression and cell motility.},
}
@article {pmid33258473,
year = {2020},
author = {Rönspies, M and Schindele, P and Puchta, H},
title = {CRISPR/Cas-mediated chromosome engineering: opening up a new avenue for plant breeding.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/eraa463},
pmid = {33258473},
issn = {1460-2431},
abstract = {The advent of powerful site-specific nucleases, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, which enables precise genome manipulation, has revolutionized plant breeding. Until recently, the main focus of researchers has been to simply knock-in or knock-out single genes, or to induce single base changes, but constant improvements of this technology have enabled more ambitious applications that aim to improve plant productivity or other desirable traits. One long-standing aim has been the induction of targeted chromosomal rearrangements (crossovers, inversions, or translocations). The feasibility of this technique has the potential to transform plant breeding, because natural rearrangements, like inversions, for example, typically present obstacles to the breeding process. In this way, genetic linkages between traits could be altered to combine or separate favorable and deleterious genes, respectively. In this review, we discuss recent breakthroughs in the field of chromosome engineering in plants and their potential applications in the field of plant breeding. In the future, these approaches might be applicable in shaping plant chromosomes in a directed manner, based on plant breeding needs.},
}
@article {pmid33258314,
year = {2020},
author = {Dhar, B and Steimberg, N and Mazzoleni, G},
title = {Point-of-Care Pathogen Detection with CRISPR-based Programmable Nucleic Acid Binding Proteins.},
journal = {ChemMedChem},
volume = {},
number = {},
pages = {},
doi = {10.1002/cmdc.202000782},
pmid = {33258314},
issn = {1860-7187},
abstract = {The contemporary discovery of extremely versatile engineered nucleic acids binding proteins has transformed a brave world in the genome-editing scientific area. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- mediated programmable nucleic acids-binding proteins have brought a revolution in the diagnostic platforms. The groundbreaking finding that bacteria and archaea harbored prokaryotes have transmitted adaptive immunity through CRISPR and CRISPR-associated (Cas) proteins has driven to revolutionize advances in molecular biology. Importantly, advances in gene editing focus how expanding visions into CRISPR-Cas biology are revolutionizing the area of molecular diagnostics for identification of DNA and RNA in emerging microbiological pathogens, Single Nucleotide Polymorphism (SNP) identifications as well as cell-free mutation. Recent advancements that include improvements in multiplexing and quantitative capabilities, and instrument-free detection of nucleic acids will potentially leverage the introduction of these novel technologies to bacterial and viral detection at the point-of-care (POC). In this review, we highlight the fundamental features of CRISPR/Cas based molecular diagnostic technologies and summarize a vision of next applications for identifications of pathogens in Point-of-care (POC) settings.},
}
@article {pmid33256602,
year = {2020},
author = {Refrégier, G and Sola, C and Guyeux, C},
title = {Unexpected diversity of CRISPR unveils some evolutionary patterns of repeated sequences in Mycobacterium tuberculosis.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {841},
pmid = {33256602},
issn = {1471-2164},
abstract = {BACKGROUND: Diversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes.<br><br>RESULTS: We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs. We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats.<br><br>CONCLUSION: This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.},
}
@article {pmid33253976,
year = {2020},
author = {Modell, AE and Siriwardena, SU and Shoba, VM and Li, X and Choudhary, A},
title = {clustered regularly interspaced short palindromic repeat-Chemical and optical control of CRISPR-associated nucleases.},
journal = {Current opinion in chemical biology},
volume = {60},
number = {},
pages = {113-121},
doi = {10.1016/j.cbpa.2020.10.003},
pmid = {33253976},
issn = {1879-0402},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system of bacteria has furnished programmable nucleases (e.g., Cas9) that are transforming the field of genome editing with applications in basic and biomedical research, biotechnology, and agriculture. However, broader real-world applications of Cas9 require precision control of its activity over dose, time, and space as off-target effects, embryonic mosaicism, chromosomal translocations, and genotoxicity have been observed with elevated and/or prolonged nuclease activity. Here, we review chemical and optical methods for precision control of Cas9's activity.},
}
@article {pmid33252243,
year = {2020},
author = {Park, KE and Frey, JF and Waters, J and Simpson, SG and Coutu, C and Plummer, S and Campbell, M and Donovan, DM and Telugu, BP},
title = {One-Step Homology Mediated CRISPR-Cas Editing in Zygotes for Generating Genome Edited Cattle.},
journal = {The CRISPR journal},
volume = {3},
number = {6},
pages = {523-534},
doi = {10.1089/crispr.2020.0047},
pmid = {33252243},
issn = {2573-1602},
abstract = {Selective breeding and genetic modification have been the cornerstone of animal agriculture. However, the current strategy of breeding animals over multiple generations to introgress novel alleles is not practical in addressing global challenges such as climate change, pandemics, and the predicted need to feed a population of 9 billion by 2050. Consequently, genome editing in zygotes to allow for seamless introgression of novel alleles is required, especially in cattle with long generation intervals. We report for the first time the use of CRISPR-Cas genome editors to introduce novel PRNP allelic variants that have been shown to provide resilience towards human prion pandemics. From one round of embryo injections, we have established six pregnancies and birth of seven edited offspring, with two founders showing >90% targeted homology-directed repair modifications. This study lays out the framework for in vitro optimization, unbiased deep-sequencing to identify editing outcomes, and generation of high frequency homology-directed repair-edited calves.},
}
@article {pmid33251765,
year = {2020},
author = {Fang, L and Wang, W and Li, G and Zhang, L and Li, J and Gan, D and Yang, J and Tang, Y and Ding, Z and Zhang, M and Zhang, W and Deng, D and Song, Z and Zhu, Q and Cui, H and Hu, Y and Chen, W},
title = {CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.},
journal = {Molecular systems biology},
volume = {16},
number = {11},
pages = {e10025},
pmid = {33251765},
issn = {1744-4292},
support = {2019SHIBS0002//Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Instiutions/ ; KQTD20180411143432337//Science, Technology and Innovation Commission of Shenzhen Municipality (Shenzhen Science and Technology Innovation Commission)/ ; JCYJ20190809154407564//Science, Technology and Innovation Commission of Shenzhen Municipality (Shenzhen Science and Technology Innovation Commission)/ ; JCYJ20180504165804015//Science, Technology and Innovation Commission of Shenzhen Municipality (Shenzhen Science and Technology Innovation Commission)/ ; 31701237//National Natural Science Foundation of China (NSFC)/ ; 31900431//National Natural Science Foundation of China (NSFC)/ ; 31970601//National Natural Science Foundation of China (NSFC)/ ; },
abstract = {Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6 A and m5 C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m5 C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non-overlapping subsets of mRNA m5 C sites and together contributed to almost all the m5 C modification in mRNA. Finally, using m1 A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.},
}
@article {pmid33251190,
year = {2020},
author = {Tavares, LM and de Jesus, LCL and da Silva, TF and Barroso, FAL and Batista, VL and Coelho-Rocha, ND and Azevedo, V and Drumond, MM and Mancha-Agresti, P},
title = {Novel Strategies for Efficient Production and Delivery of Live Biotherapeutics and Biotechnological Uses of Lactococcus lactis: The Lactic Acid Bacterium Model.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {517166},
pmid = {33251190},
issn = {2296-4185},
abstract = {Lactic acid bacteria (LAB) are traditionally used in fermentation and food preservation processes and are recognized as safe for consumption. Recently, they have attracted attention due to their health-promoting properties; many species are already widely used as probiotics for treatment or prevention of various medical conditions, including inflammatory bowel diseases, infections, and autoimmune disorders. Some LAB, especially Lactococcus lactis, have been engineered as live vehicles for delivery of DNA vaccines and for production of therapeutic biomolecules. Here, we summarize work on engineering of LAB, with emphasis on the model LAB, L. lactis. We review the various expression systems for the production of heterologous proteins in Lactococcus spp. and its use as a live delivery system of DNA vaccines and for expression of biotherapeutics using the eukaryotic cell machinery. We have included examples of molecules produced by these expression platforms and their application in clinical disorders. We also present the CRISPR-Cas approach as a novel methodology for the development and optimization of food-grade expression of useful substances, and detail methods to improve DNA delivery by LAB to the gastrointestinal tract. Finally, we discuss perspectives for the development of medical applications of recombinant LABs involving animal model studies and human clinical trials, and we touch on the main safety issues that need to be taken into account so that bioengineered versions of these generally recognized as safe organisms will be considered acceptable for medical use.},
}
@article {pmid33251158,
year = {2020},
author = {Kumar, P and Malik, YS and Ganesh, B and Rahangdale, S and Saurabh, S and Natesan, S and Srivastava, A and Sharun, K and Yatoo, MI and Tiwari, R and Singh, RK and Dhama, K},
title = {CRISPR-Cas System: An Approach With Potentials for COVID-19 Diagnosis and Therapeutics.},
journal = {Frontiers in cellular and infection microbiology},
volume = {10},
number = {},
pages = {576875},
pmid = {33251158},
issn = {2235-2988},
mesh = {Animals ; COVID-19/diagnosis/therapy/*virology ; *CRISPR-Cas Systems ; Humans ; SARS-CoV-2/*genetics/metabolism ; },
abstract = {COVID-19, the human coronavirus disease caused by SARS-CoV-2, was reported for the first time in Wuhan, China in late 2019. COVID-19 has no preventive vaccine or proven standard pharmacological treatment, and consequently, the outbreak swiftly became a pandemic affecting more than 215 countries around the world. For the diagnosis of COVID-19, the only reliable diagnostics is a qPCR assay. Among other diagnostic tools, the CRISPR-Cas system is being investigated for rapid and specific diagnosis of COVID-19. The CRISPR-Cas-based methods diagnose the SARS-CoV-2 infections within an hour. Apart from its diagnostic ability, CRISPR-Cas system is also being assessed for antiviral therapy development; however, till date, no CRISPR-based therapy has been approved for human use. The Prophylactic Antiviral CRISPR in huMAN cells (PAC-MAN), which is Cas 13 based strategy, has been developed against coronavirus. Although this strategy has the potential to be developed as a therapeutic modality, it may face significant challenges for approval in human clinical trials. This review is focused on describing potential use and challenges of CRISPR-Cas based approaches for the development of rapid and accurate diagnostic technique and/or a possible therapeutic alternative for combating COVID-19. The assessment of potential risks associated with use of CRISPR will be important for future clinical advancements.},
}
@article {pmid33250920,
year = {2020},
author = {Alcorta, J and Alarcón-Schumacher, T and Salgado, O and Díez, B},
title = {Taxonomic Novelty and Distinctive Genomic Features of Hot Spring Cyanobacteria.},
journal = {Frontiers in genetics},
volume = {11},
number = {},
pages = {568223},
pmid = {33250920},
issn = {1664-8021},
abstract = {Several cyanobacterial species are dominant primary producers in hot spring microbial mats. To date, hot spring cyanobacterial taxonomy, as well as the evolution of their genomic adaptations to high temperatures, are poorly understood, with genomic information currently available for only a few dominant genera, including Fischerella and Synechococcus. To address this knowledge gap, the present study expands the genomic landscape of hot spring cyanobacteria and traces the phylum-wide genomic consequences of evolution in high temperature environments. From 21 globally distributed hot spring metagenomes, with temperatures between 32 and 75°C, 57 medium- and high-quality cyanobacterial metagenome-assembled genomes were recovered, representing taxonomic novelty for 1 order, 3 families, 15 genera and 36 species. Comparative genomics of 93 hot spring genomes (including the 57 metagenome-assembled genomes) and 66 non-thermal genomes, showed that the former have smaller genomes and a higher GC content, as well as shorter proteins that are more hydrophilic and basic, when compared to the non-thermal genomes. Additionally, the core accessory orthogroups from the hot spring genomes of some genera had a greater abundance of functional categories, such as inorganic ion metabolism, translation and post-translational modifications. Moreover, hot spring genomes showed increased abundances of inorganic ion transport and amino acid metabolism, as well as less replication and transcription functions in the protein coding sequences. Furthermore, they showed a higher dependence on the CRISPR-Cas defense system against exogenous nucleic acids, and a reduction in secondary metabolism biosynthetic gene clusters. This suggests differences in the cyanobacterial response to environment-specific microbial communities. This phylum-wide study provides new insights into cyanobacterial genomic adaptations to a specific niche where they are dominant, which could be essential to trace bacterial evolution pathways in a warmer world, such as the current global warming scenario.},
}
@article {pmid33249867,
year = {2020},
author = {Kumar, P and Sinha, R and Shukla, P},
title = {Artificial intelligence and synthetic biology approaches for human gut microbiome.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10408398.2020.1850415},
pmid = {33249867},
issn = {1549-7852},
abstract = {The gut microbiome comprises a variety of microorganisms whose genes encode proteins to carry out crucial metabolic functions that are responsible for the majority of health-related issues in human beings. The advent of the technological revolution in artificial intelligence (AI) assisted synthetic biology (SB) approaches will play a vital role in the modulating the therapeutic and nutritive potential of probiotics. This can turn human gut as a reservoir of beneficial bacterial colonies having an immense role in immunity, digestion, brain function, and other health benefits. Hence, in the present review, we have discussed the role of several gene editing tools and approaches in synthetic biology that have equipped us with novel tools like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) systems to precisely engineer probiotics for diagnostic, therapeutic and nutritive value. A brief discussion over the AI techniques to understand the metagenomic data from the healthy and diseased gut microbiome is also presented. Further, the role of AI in potentially impacting the pace of developments in SB and its current challenges is also discussed. The review also describes the health benefits conferred by engineered microbes through the production of biochemicals, nutraceuticals, drugs or biotherapeutics molecules etc. Finally, the review concludes with the challenges and regulatory concerns in adopting synthetic biology engineered microbes for clinical applications. Thus, the review presents a synergistic approach of AI and SB toward human gut microbiome for better health which will provide interesting clues to researchers working in the area of rapidly evolving food and nutrition science.},
}
@article {pmid33248248,
year = {2020},
author = {Rabinowitz, R and Offen, D},
title = {Single-Base Resolution: Increasing the Specificity of the CRISPR-Cas System in Gene Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2020.11.009},
pmid = {33248248},
issn = {1525-0024},
abstract = {The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. The Cas protein, an RNA-guided programmable nuclease, generates a double-strand break at precise genomic loci. However, the use of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system to distinguish between single-nucleotide variations is challenging. The promiscuity of the guide RNA (gRNA) and its mismatch tolerance make allele-specific targeting an elusive goal. This review presents a meta-analysis of previous studies reporting position-dependent mismatch tolerance within the gRNA. We also examine the conservativity of the seed sequence, a region within the gRNA with stringent sequence dependency, and propose the existence of a subregion within the seed sequence with a higher degree of specificity. In addition, we summarize the reports on high-fidelity Cas nucleases with improved specificity and compare the standard gRNA design methodology to the single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach, an alternative method for allele-specific targeting. The combination of the two methods may be advantageous in designing CRISPR-based therapeutics and diagnostics for heterozygous patients.},
}
@article {pmid33248026,
year = {2020},
author = {McBride, TM and Schwartz, EA and Kumar, A and Taylor, DW and Fineran, PC and Fagerlund, RD},
title = {Diverse CRISPR-Cas Complexes Require Independent Translation of Small and Large Subunits from a Single Gene.},
journal = {Molecular cell},
volume = {80},
number = {6},
pages = {971-979.e7},
doi = {10.1016/j.molcel.2020.11.003},
pmid = {33248026},
issn = {1097-4164},
abstract = {CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select systems, multiple CRISPR-Cas types remain uncharacterized. Here, we investigated the previously uncharacterized type I-D interference complex and revealed that it is a genetic and structural hybrid with similarity to both type I and type III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across diverse type I-D, I-B, and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution.},
}
@article {pmid33247091,
year = {2020},
author = {Mancuso, P and Chen, C and Kaminski, R and Gordon, J and Liao, S and Robinson, JA and Smith, MD and Liu, H and Sariyer, IK and Sariyer, R and Peterson, TA and Donadoni, M and Williams, JB and Siddiqui, S and Bunnell, BA and Ling, B and MacLean, AG and Burdo, TH and Khalili, K},
title = {CRISPR based editing of SIV proviral DNA in ART treated non-human primates.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {6065},
pmid = {33247091},
issn = {2041-1723},
support = {U42 OD024282/OD/NIH HHS/United States ; P51 OD011104/OD/NIH HHS/United States ; R01 MH116844/MH/NIMH NIH HHS/United States ; U42 OD010568/OD/NIH HHS/United States ; R01 NS104016/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Anti-Retroviral Agents/*pharmacology ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; DNA, Viral/blood/*genetics ; *Gene Editing ; Genome, Viral ; Humans ; Lung/drug effects/virology ; Lymph Nodes/drug effects/virology ; Macaca mulatta ; Proviruses/drug effects/*genetics ; Simian Acquired Immunodeficiency Syndrome/blood/virology ; Simian Immunodeficiency Virus/*genetics ; Spleen/pathology/virology ; Tissue Distribution ; Transgenes ; },
abstract = {Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.},
}
@article {pmid33245685,
year = {2020},
author = {Vatankhah, M and Azizi, A and Sanajouyan Langeroudi, A and Ataei Azimi, S and Khorsand, I and Kerachian, MA and Motaei, J},
title = {CRISPR-based biosensing systems: a way to rapidly diagnose COVID-19.},
journal = {Critical reviews in clinical laboratory sciences},
volume = {},
number = {},
pages = {1-27},
doi = {10.1080/10408363.2020.1849010},
pmid = {33245685},
issn = {1549-781X},
abstract = {The outbreak of the emerging SARS-CoV-2 virus has highlighted the challenges of detecting viral infections, especially in resource-limited settings. The SARS-CoV-2 virus transmission chain is interrupted when screening and diagnosis can be performed on a large scale by identifying asymptomatic or moderately symptomatic patients. Diagnosis of COVID-19 with reverse transcription polymerase chain reaction (RT-PCR) has been limited due to inadequate access to complex, expensive equipment and reagents, which has impeded efforts to reduce the spread of virus transmission. Recently, the development of several diagnostic platforms based on the CRISPR-Cas system has reduced the dependence on RT-PCR. The first CRISPR-based diagnostic test for SARS-CoV-2 was recently approved by the U.S. Food and Drug Administration. The biosensing systems have several important features that make them suitable for point-of-care tests, including the speed of design and synthesis of each platform in less than a few days, an assay time of 1-2 h, and the cost of materials and reagents less than one dollar per test. The HUDSON-SHERLOCK and STOPCovid biosensing systems, as field-deployable and rapid diagnostic tests, can detect low-copy viruses in body fluids without nucleic acid extraction and with minimal equipment. In addition, Cas13-based treatment strategies could potentially be an effective antiviral strategy for the prevention and treatment of emerging pandemic viruses such as SARS-CoV-2. In this review, we describe recent advances in CRISPR-based diagnostic platforms with an emphasis on their use in the rapid diagnosis and potential treatment of COVID-19.},
}
@article {pmid33244929,
year = {2020},
author = {Zhang, D and Luo, Y and Chen, W},
title = {[Current development of gene editing].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {11},
pages = {2345-2356},
doi = {10.13345/j.cjb.190557},
pmid = {33244929},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; China ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Plants/genetics ; },
abstract = {As the breakthrough in gene editing, represented by CRISPR/Cas9, gene manipulations now are more maneuverable, economically feasible and time saving. It is possible for China to catch an overtaking in researching and industrializing of downside sections (especially the application of plant gene editing), also the incubation of professional companies in gene editing fields. For this consideration, it is necessary and urgent to find the key demands and potential application for gene editing in China. Questionnaire and statistic analysis were carried out to find the key demands and the most potential application fields of the development for gene editing. Firstly, an ordered multi-classification Logistic regression model was established following with dependent variable analysis. Eight out of 24 questionnaires questions in 4 categories were regarded as independent variables with significance test. Then, regression model based on ordered multi-classification logistic method was established to analyze the specific impact of different options on the development of gene editing. The results showed that most researchers in the field of gene editing take the view that development of potential competitive advantages lies in the field of plant science. The results also showed that major gene editing experts believe more attention should be paid on how to carry out technology industrialization while focusing on basic technology development, as well as the development of potential competitive advantages of gene editing technology in plant field. To promote the development of gene editing in China, not only the participation of scientific research institution was needed, but also the synergy of various forces both universities and governments. It is urgent both properly guiding public opinion on gene editing and establishing a national safety standard system. At the same time, the key point of technology risk avoidance should be put on biological weapons and bioterrorism, gene editing related infectious disease, and the potential risk of species genetic change on the ecological environment, etc.},
}
@article {pmid33242354,
year = {2020},
author = {Sharma, B and Shukla, P},
title = {Futuristic avenues of metabolic engineering techniques in bioremediation.},
journal = {Biotechnology and applied biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/bab.2080},
pmid = {33242354},
issn = {1470-8744},
support = {BT/PR27437/BCE/8/1433/2018//Department of Biotechnology, Government of India/ ; SR/FT/LS-31/2012//Department of Science and Technology, Government of India/ ; 1196 SR/FST/LS-I/2017/4//Department of Science and Technology, Government of India/ ; },
abstract = {Bioremediation is a promising technology for the treatment of environmental contaminants and paving new avenues for the betterment of the environment. Over the last some years, several approaches have been employed to optimize the genetic machinery of microorganisms relevant to bioremediation. Metabolic engineering is one of them that provides a new insight for bioremediation. This review envisages the critical role of these techniques toward exploring the possibilities of the creation of a new pathway, leading to pathway expansion to new substrates by assembling of catabolic modules from different origins in the same microbial cell. The recombinant DNA technology and gene editing tools were also explored for the construction of metabolically engineered microbial strains for the degradation of complex pollutants. Moreover, the importance of CRISPR-Cas system for knock-in and knock-out of genes was described by using recent studies. Further, the idea of the cocultivation of more than one metabolic engineered microbial communities is also discussed, which can be crucial in the bioremediation of multiple and complex pollutants. Finally, this review also elucidates the effective application of metabolic engineering in bioremediation through these techniques and tools.},
}
@article {pmid33239638,
year = {2020},
author = {Manav, MC and Van, LB and Lin, J and Fuglsang, A and Peng, X and Brodersen, DE},
title = {Structural basis for inhibition of an archaeal CRISPR-Cas type I-D large subunit by an anti-CRISPR protein.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5993},
pmid = {33239638},
issn = {2041-1723},
mesh = {Archaeal Proteins/*antagonists &amp; inhibitors/metabolism/ultrastructure ; CRISPR-Associated Proteins/*antagonists &amp; inhibitors/metabolism/ultrastructure ; CRISPR-Cas Systems/genetics ; DNA Cleavage ; Host-Pathogen Interactions/genetics ; Protein Domains/genetics ; Repressor Proteins/genetics/*metabolism ; Rudiviridae/genetics/metabolism/pathogenicity ; Sulfolobus/*genetics/virology ; Viral Proteins/genetics/*metabolism/ultrastructure ; },
abstract = {A hallmark of type I CRISPR-Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3' subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR-Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.},
}
@article {pmid33238709,
year = {2020},
author = {Pang, B and Xu, J and Liu, Y and Peng, H and Feng, W and Cao, Y and Wu, J and Xiao, H and Pabbaraju, K and Tipples, G and Joyce, MA and Saffran, HA and Tyrrell, DL and Zhang, H and Le, XC},
title = {Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology.},
journal = {Analytical chemistry},
volume = {92},
number = {24},
pages = {16204-16212},
doi = {10.1021/acs.analchem.0c04047},
pmid = {33238709},
issn = {1520-6882},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Humans ; *Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/*genetics ; },
abstract = {We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30-39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.},
}
@article {pmid33237723,
year = {2020},
author = {Paßreiter, A and Thomas, A and Grogna, N and Delahaut, P and Thevis, M},
title = {First Steps toward Uncovering Gene Doping with CRISPR/Cas by Identifying SpCas9 in Plasma via HPLC-HRMS/MS.},
journal = {Analytical chemistry},
volume = {92},
number = {24},
pages = {16322-16328},
doi = {10.1021/acs.analchem.0c04445},
pmid = {33237723},
issn = {1520-6882},
abstract = {The discovery of the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system as a programmable, RNA-guided endonuclease has revolutionized the utilization of gene technology. Because it enables the precise modification of any desired DNA sequence and surpasses all hitherto existing alternatives for gene editing in many ways, it is one of the most frequently used tools for genome editing. However, these advantages also potentially facilitate the illicit use of the CRISPR/Cas system in order to achieve performance-enhancing effects in sporting competitions. This abuse is classified as gene doping, which is banned in sports according to the Prohibited List of the World Anti-Doping Agency (WADA). Therefore, there is a pressing need for an adequate analytical method to detect the misuse of the CRISPR/Cas system by athletes. Hence, the first aim accomplished with this study was the identification of the exogenous protein Cas9 from the bacterium Streptococcus pyogenes (SpCas9) in plasma samples by means of a bottom-up analytical approach via immunoaffinity purification, tryptic digestion, and subsequent detection by HPLC-HRMS/MS. A qualitative method validation was conducted with three specific peptides allowing for a limit of detection of 25 ng/mL. Additionally, it was shown that the developed method is also applicable to the detection of (illicit) gene regulation through the identification of catalytically inactive Cas9. A proof-of-concept administration study employing an in vivo mouse model revealed a detection window of SpCas9 for up to 8 h post administration, confirming the suitability of the test strategy for the analysis of authentic doping control samples.},
}
@article {pmid33237352,
year = {2020},
author = {Biagioni, A and Laurenzana, A and Menicacci, B and Peppicelli, S and Andreucci, E and Bianchini, F and Guasti, D and Paoli, P and Serratì, S and Mocali, A and Calorini, L and Del Rosso, M and Fibbi, G and Chillà, A and Margheri, F},
title = {uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00018-020-03707-4},
pmid = {33237352},
issn = {1420-9071},
abstract = {Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR-Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR-EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.},
}
@article {pmid33235312,
year = {2020},
author = {Hunziker, J and Nishida, K and Kondo, A and Kishimoto, S and Ariizumi, T and Ezura, H},
title = {Multiple gene substitution by Target-AID base-editing technology in tomato.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {20471},
pmid = {33235312},
issn = {2045-2322},
abstract = {The use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes, SlDDB1, SlDET1 and SlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T0 lines, we obtained 10 independent T0 lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.},
}
@article {pmid33235250,
year = {2020},
author = {Kumar, S and Punetha, M and Jose, B and Bharati, J and Khanna, S and Sonwane, A and Green, JA and Whitworth, K and Sarkar, M},
title = {Modulation of granulosa cell function via CRISPR-Cas fuelled editing of BMPR-IB gene in goats (Capra hircus).},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {20446},
pmid = {33235250},
issn = {2045-2322},
abstract = {BMPs are multifunctional growth factors implicated in regulating the ovarian function as key intra-ovarian factors. Biological effects of BMPs are mediated through binding with membrane bound receptors like BMPR-IB and initiating downstream Smad signaling pathway. FecB mutation, regarded as a loss of function mutation in the BMPR-IB gene was identified in certain sheep breeds having high fecundity. Similar type of fecundity genes in goats have not been discovered so far. Hence, the current study was designed to investigate the effects of BMPR-IB gene modulation on granulosa cell function in goats. The BMPR-IB gene was knocked out using CRISPR-Cas technology in granulosa cells and cultured in vitro with BMP-4 stimulation for three different durations In addition, the FecB mutation was introduced in the BMPR-IB gene applying Easi-CRISPR followed by BMP-4/7 stimulation for 72 h. Steroidogenesis and cell viability were studied to explore the granulosa cell function on BMPR-IB gene modulation. BMPRs were found to be expressed stage specifically in granulosa cells of goats. Higher transcriptional abundance of R-Smads, LHR and FSHR indicating sensitisation of Smad signaling and increased gonadotropin sensitivity along with a significant reduction in the cell proliferation and viability was observed in granulosa cells upon BMPR-IB modulation. The inhibitory action of BMP-4/7 on P4 secretion was abolished in both KO and KI cells. Altogether, the study has revealed an altered Smad signaling, steroidogenesis and cell viability upon modulation of BMPR-IB gene in granulosa cells similar to that are documented in sheep breeds carrying the FecB mutation.},
}
@article {pmid33233548,
year = {2020},
author = {Ghribi, M and Nouemssi, SB and Meddeb-Mouelhi, F and Desgagné-Penix, I},
title = {Genome Editing by CRISPR-Cas: A Game Change in the Genetic Manipulation of Chlamydomonas.},
journal = {Life (Basel, Switzerland)},
volume = {10},
number = {11},
pages = {},
pmid = {33233548},
issn = {2075-1729},
support = {950-232164//Canada Research Chairs/ ; IT12310//Mitacs/ ; IT13619//Mitacs/ ; },
abstract = {Microalgae are promising photosynthetic unicellular eukaryotes among the most abundant on the planet and are considered as alternative sustainable resources for various industrial applications. Chlamydomonas is an emerging model for microalgae to be manipulated by multiple biotechnological tools in order to produce high-value bioproducts such as biofuels, bioactive peptides, pigments, nutraceuticals, and medicines. Specifically, Chlamydomonas reinhardtii has become a subject of different genetic-editing techniques adapted to modulate the production of microalgal metabolites. The main nuclear genome-editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and more recently discovered the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) nuclease system. The latter, shown to have an interesting editing capacity, has become an essential tool for genome editing. In this review, we highlight the available literature on the methods and the applications of CRISPR-Cas for C. reinhardtii genetic engineering, including recent transformation methods, most used bioinformatic tools, best strategies for the expression of Cas protein and sgRNA, the CRISPR-Cas mediated gene knock-in/knock-out strategies, and finally the literature related to CRISPR expression and modification approaches.},
}
@article {pmid33232480,
year = {2020},
author = {Basar, R and Daher, M and Rezvani, K},
title = {Next-generation cell therapies: the emerging role of CAR-NK cells.},
journal = {Blood advances},
volume = {4},
number = {22},
pages = {5868-5876},
pmid = {33232480},
issn = {2473-9537},
support = {P01 CA148600/CA/NCI NIH HHS/United States ; P30 CA016672/CA/NCI NIH HHS/United States ; P50 CA100632/CA/NCI NIH HHS/United States ; R01 CA211044/CA/NCI NIH HHS/United States ; },
abstract = {T cells engineered with chimeric antigen receptors (CARs) have revolutionized the field of cell therapy and changed the paradigm of treatment for many patients with relapsed or refractory B-cell malignancies. Despite this progress, there are limitations to CAR-T cell therapy in both the autologous and allogeneic settings, including practical, logistical, and toxicity issues. Given these concerns, there is a rapidly growing interest in natural killer cells as alternative vehicles for CAR engineering, given their unique biological features and their established safety profile in the allogeneic setting. Other immune effector cells, such as invariant natural killer T cells, γδ T cells, and macrophages, are attracting interest as well and eventually may be added to the repertoire of engineered cell therapies against cancer. The pace of these developments will undoubtedly benefit from multiple innovative technologies, such as the CRISPR-Cas gene editing system, which offers great potential to enhance the natural ability of immune effector cells to eliminate refractory cancers.},
}
@article {pmid33230293,
year = {2020},
author = {Vo, PLH and Ronda, C and Klompe, SE and Chen, EE and Acree, C and Wang, HH and Sternberg, SH},
title = {CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41587-020-00745-y},
pmid = {33230293},
issn = {1546-1696},
support = {MCB-1453219//National Science Foundation (NSF)/ ; PATH1016691//Burroughs Wellcome Fund (BWF)/ ; },
abstract = {Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.},
}
@article {pmid33230133,
year = {2020},
author = {O'Brien, RE and Santos, IC and Wrapp, D and Bravo, JPK and Schwartz, EA and Brodbelt, JS and Taylor, DW},
title = {Structural basis for assembly of non-canonical small subunits into type I-C Cascade.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5931},
pmid = {33230133},
issn = {2041-1723},
support = {R01 GM121714/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/metabolism ; Binding Sites ; CRISPR-Associated Proteins/*chemistry/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryoelectron Microscopy ; DNA/chemistry/metabolism ; Desulfovibrio vulgaris/chemistry/genetics ; Models, Molecular ; Multiprotein Complexes/chemistry/metabolism ; Nucleotide Motifs ; Protein Conformation ; Protein Subunits/chemistry/metabolism ; RNA, Bacterial/chemistry/metabolism ; },
abstract = {Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.},
}
@article {pmid33226067,
year = {2020},
author = {Padilha, VA and Alkhnbashi, OS and Tran, VD and Shah, SA and L F Carvalho, ACP and Backofen, R},
title = {Casboundary: Automated definition of integral Cas cassettes.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btaa984},
pmid = {33226067},
issn = {1367-4811},
abstract = {MOTIVATION: CRISPR-Cas are important systems found in most archaeal and many bacterial genomes, providing adaptive immunity against mobile genetic elements in prokaryotes. The CRISPR-Cas systems are encoded by a set of consecutive cas genes, here termed cassette. The identification of cassette boundaries is key for finding cassettes in CRISPR research field. This is often carried out by using Hidden Markov Models and manual annotation. In this paper, we propose the first method able to automatically define the cassette boundaries. In addition, we present a Cas type predictive model used by the method to assign each gene located in the region defined by a cassette's boundaries a Cas label from a set of pre-defined Cas types. Furthermore, the proposed method can detect potentially new cas genes and decompose a cassette into its modules.<br><br>RESULTS: We evaluate the predictive performance of our proposed method on data collected from the two most recent CRISPR classification studies. In our experiments, we obtain an average similarity of 0.86 between the predicted and expected cassettes. Besides, we achieve F-scores above 0.9 for the classification of cas genes of known types and 0.73 for the unknown ones. Finally, we conduct two additional study cases, where we investigate the occurrence of potentially new cas genes and the occurrence of module exchange between different genomes.<br><br>AVAILABILITY: https://github.com/BackofenLab/Casboundary.},
}
@article {pmid33226023,
year = {2020},
author = {Laoharawee, K and Johnson, MJ and Lahr, WS and Peterson, JJ and Webber, BR and Moriarity, BS},
title = {Genome Engineering of Primary Human B Cells Using CRISPR/Cas9.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {165},
pages = {},
doi = {10.3791/61855},
pmid = {33226023},
issn = {1940-087X},
support = {R01 AI146009/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, CD19/metabolism ; B-Lymphocytes/cytology/*metabolism ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Proliferation ; Cells, Cultured ; Dependovirus/metabolism ; Genetic Engineering/*methods ; *Genome, Human ; Green Fluorescent Proteins/metabolism ; Humans ; INDEL Mutation/genetics ; },
abstract = {B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function-to produce large amounts of antibodies-can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.},
}
@article {pmid33219687,
year = {2020},
author = {Nobrega, FL and Walinga, H and Dutilh, BE and Brouns, SJJ},
title = {Prophages are associated with extensive CRISPR-Cas auto-immunity.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12074-12084},
doi = {10.1093/nar/gkaa1071},
pmid = {33219687},
issn = {1362-4962},
abstract = {CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR-Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100 000 bacterial genomes. We found STS in all CRISPR-Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR-Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR-Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects as mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages.},
}
@article {pmid33218076,
year = {2020},
author = {Tanmoy, AM and Saha, C and Sajib, MSI and Saha, S and Komurian-Pradel, F and van Belkum, A and Louwen, R and Saha, SK and Endtz, HP},
title = {CRISPR-Cas Diversity in Clinical Salmonella enterica Serovar Typhi Isolates from South Asian Countries.},
journal = {Genes},
volume = {11},
number = {11},
pages = {},
pmid = {33218076},
issn = {2073-4425},
abstract = {Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a global health concern and its treatment is problematic due to the rise in antimicrobial resistance (AMR). Rapid detection of patients infected with AMR positive S. Typhi is, therefore, crucial to prevent further spreading. Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas), is an adaptive immune system that initially was used for typing purposes. Later, it was discovered to play a role in defense against phages and plasmids, including ones that carry AMR genes, and, at present, it is being explored for its usage in diagnostics. Despite the availability of whole-genome sequences (WGS), very few studied the CRISPR-Cas system of S. Typhi, let alone in typing purposes or relation to AMR. In the present study, we analyzed the CRISPR-Cas system of S. Typhi using WGS data of 1059 isolates obtained from Bangladesh, India, Nepal, and Pakistan in combination with demographic data and AMR status. Our results reveal that the S. Typhi CRISPR loci can be classified into two groups: A (evidence level >2) and B (evidence level ≤2), in which we identified a total of 47 unique spacers and 15 unique direct repeats. Further analysis of the identified spacers and repeats demonstrated specific patterns that harbored significant associations with genotype, demographic characteristics, and AMR status, thus raising the possibility of their usage as biomarkers. Potential spacer targets were identified and, interestingly, the phage-targeting spacers belonged to the group-A and plasmid-targeting spacers to the group-B CRISPR loci. Further analyses of the spacer targets led to the identification of an S. Typhi protospacer adjacent motif (PAM) sequence, TTTCA/T. New cas-genes known as DinG, DEDDh, and WYL were also discovered in the S. Typhi genome. However, a specific variant of the WYL gene was only identified in the extensively drug-resistant (XDR) lineage from Pakistan and ciprofloxacin-resistant lineage from Bangladesh. From this work, we conclude that there are strong correlations between variations identified in the S. Typhi CRISPR-Cas system and endemic AMR positive S. Typhi isolates.},
}
@article {pmid33217332,
year = {2020},
author = {Münch, PC and Franzosa, EA and Stecher, B and McHardy, AC and Huttenhower, C},
title = {Identification of Natural CRISPR Systems and Targets in the Human Microbiome.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2020.10.010},
pmid = {33217332},
issn = {1934-6069},
support = {U54 DK102557/DK/NIDDK NIH HHS/United States ; },
abstract = {Many bacteria resist invasive DNA by incorporating sequences into CRISPR loci, which enable sequence-specific degradation. CRISPR systems have been well studied from isolate genomes, but culture-independent metagenomics provide a new window into their diversity. We profiled CRISPR loci and cas genes in the body-wide human microbiome using 2,355 metagenomes, yielding functional and taxonomic profiles for 2.9 million spacers by aligning the spacer content to each sample's metagenome and corresponding gene families. Spacer and repeat profiles agree qualitatively with those from isolate genomes but expand their diversity by approximately 13-fold, with the highest spacer load present in the oral microbiome. The taxonomy of spacer sequences parallels that of their source community, with functional targets enriched for viral elements. When coupled with cas gene systems, CRISPR-Cas subtypes are highly site and taxon specific. Our analysis provides a comprehensive collection of natural CRISPR-cas loci and targets in the human microbiome.},
}
@article {pmid33216174,
year = {2020},
author = {Gao, Y and Wu, S and Pan, J and Zhang, K and Li, X and Xu, Y and Jin, C and He, X and Shi, J and Ma, L and Wu, F and Yao, Y and Wang, P and He, Q and Lan, F and Zhang, H and Tian, M},
title = {CRISPR/Cas9-edited triple-fusion reporter gene imaging of dynamics and function of transplanted human urinary-induced pluripotent stem cell-derived cardiomyocytes.},
journal = {European journal of nuclear medicine and molecular imaging},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00259-020-05087-0},
pmid = {33216174},
issn = {1619-7089},
support = {81761148029, 81725009, 81900255//National Natural Science Foundation of China/ ; },
abstract = {PURPOSE: To investigate the post-transplantation behaviour and therapeutic efficacy of human urinary-induced pluripotent stem cell-derived cardiomyocytes (hUiCMs) in infarcted heart.<br><br>METHODS: We used clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology to integrate a triple-fusion (TF) reporter gene into the AAVS1 locus in human urine-derived hiPSCs (hUiPSCs) to generate TF-hUiPSCs that stably expressed monomeric red fluorescent protein for fluorescence imaging, firefly luciferase for bioluminescence imaging (BLI) and herpes simplex virus thymidine kinase for positron emission tomography (PET) imaging.<br><br>RESULTS: Transplanted cardiomyocytes derived from TF-hUiPSCs (TF-hUiCMs) engrafted and proliferated in the infarcted heart as monitored by both BLI and PET imaging and significantly improved cardiac function. Under ischaemic conditions, TF-hUiCMs enhanced cardiomyocyte (CM) glucose metabolism and promoted angiogenic activity.<br><br>CONCLUSION: This study established a CRISPR/Cas9-mediated multimodality reporter gene imaging system that can determine the dynamics and function of TF-hUiCMs in myocardial infarction, which is helpful for investigating the application of stem cell therapy.},
}
@article {pmid33213345,
year = {2020},
author = {Ray, SK and Mukherjee, S},
title = {Genome editing with CRISPR-Cas9: A budding biological contrivance for colorectal carcinoma research and its perspective in molecular medicine.},
journal = {Current molecular medicine},
volume = {},
number = {},
pages = {},
doi = {10.2174/1566524020666201119143943},
pmid = {33213345},
issn = {1875-5666},
abstract = {Genome editing is an addition, deletion, or replacement of a gene for wiping out or initiating explicit and preferred characters in the genome. Utilizing gene editing tools like CRISPR-Cas9 technology could be accomplished either by gene-based methodology or protein based technology that has been under scrutiny for protracted time wherein physical techniques, viral and non-viral strategies have been utilized together. Transplanting ex vivo CRISPR edited cells empowers screening of single guide RNAs with high-throughput and CRISPR based screening in organoids transplantation to validate cancer cells including colorectal carcinoma in various phases of its development and treatment. CRISPR knockout screens have recognized genes driving an interest in the colon cancer develop hallmarks, outstandingly for the disclosure of drug resistance mechanism in some cancer cell lines with single guide RNA. A benefit of this approach is to deal with genomic screening of CRISPR knockout, disrupts gene expression, rather than the partial knockdown which are frequently accomplished with RNA interference and CRISPR-Cas technology. Due to its proficient editing of the target gene, along with CRISPR/Cas system, this technique is used in the treatment of diverse types of cancer. In recent time research showed that CRISPR/Cas gene editing tool potentially reformed expression of long non-coding RNA in colorectal carcinoma. CRISPR/Cas9 technology will positively fuel the advancement of further in vivo gene editing clinical trials in colon cancer for forthcoming days and will have an immense impact in molecular medicine.},
}
@article {pmid33208517,
year = {2020},
author = {Bourgeois, J and Lazinski, DW and Camilli, A},
title = {Identification of Spacer and Protospacer Sequence Requirements in the Vibrio cholerae Type I-E CRISPR/Cas System.},
journal = {mSphere},
volume = {5},
number = {6},
pages = {},
pmid = {33208517},
issn = {2379-5042},
support = {T32 GM007310/GM/NIGMS NIH HHS/United States ; R37 AI055058/AI/NIAID NIH HHS/United States ; R01 AI055058/AI/NIAID NIH HHS/United States ; T32 GM008448/GM/NIGMS NIH HHS/United States ; R21 AI147658/AI/NIAID NIH HHS/United States ; },
abstract = {The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5'-TT-3' protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5'-TT-3' PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5' position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5'-RTT-3' PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5'-RTT-3' PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5' spacer pyrimidine.IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.},
}
@article {pmid33198099,
year = {2020},
author = {Shelenkov, A and Petrova, L and Fomina, V and Zamyatin, M and Mikhaylova, Y and Akimkin, V},
title = {Multidrug-Resistant Proteus mirabilis Strain with Cointegrate Plasmid.},
journal = {Microorganisms},
volume = {8},
number = {11},
pages = {},
pmid = {33198099},
issn = {2076-2607},
support = {075-15-2019-1666//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Proteus mirabilis is a component of the normal intestinal microflora of humans and animals, but can cause urinary tract infections and even sepsis in hospital settings. In recent years, the number of multidrug-resistant P. mirabilis isolates, including the ones producing extended-spectrum β-lactamases (ESBLs), is increasing worldwide. However, the number of investigations dedicated to this species, especially, whole-genome sequencing, is much lower in comparison to the members of the ESKAPE pathogens group. This study presents a detailed analysis of clinical multidrug-resistant ESBL-producing P. mirabilis isolate using short- and long-read whole-genome sequencing, which allowed us to reveal possible horizontal gene transfer between Klebsiella pneumoniae and P. mirabilis plasmids and to locate the CRISPR-Cas system in the genome together with its probable phage targets, as well as multiple virulence genes. We believe that the data presented will contribute to the understanding of antibiotic resistance acquisition and virulence mechanisms for this important pathogen.},
}
@article {pmid33196849,
year = {2020},
author = {Chai, P and Yu, J and Jia, R and Wen, X and Ding, T and Zhang, X and Ni, H and Jia, R and Ge, S and Zhang, H and Fan, X},
title = {Generation of onco-enhancer enhances chromosomal remodeling and accelerates tumorigenesis.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12135-12150},
doi = {10.1093/nar/gkaa1051},
pmid = {33196849},
issn = {1362-4962},
abstract = {Chromatin remodeling impacts the structural neighborhoods and regulates gene expression. However, the role of enhancer-guided chromatin remodeling in the gene regulation remains unclear. Here, using RNA-seq and ChIP-seq, we identified for the first time that neurotensin (NTS) serves as a key oncogene in uveal melanoma and that CTCF interacts with the upstream enhancer of NTS and orchestrates an 800 kb chromosomal loop between the promoter and enhancer. Intriguingly, this novel CTCF-guided chromatin loop was ubiquitous in a cohort of tumor patients. In addition, a disruption in this chromosomal interaction prevented the histone acetyltransferase EP300 from embedding in the promoter of NTS and resulted in NTS silencing. Most importantly, in vitro and in vivo experiments showed that the ability of tumor formation was significantly suppressed via deletion of the enhancer by CRISPR-Cas9. These studies delineate a novel onco-enhancer guided epigenetic mechanism and provide a promising therapeutic concept for disease therapy.},
}
@article {pmid33195154,
year = {2020},
author = {Malcı, K and Walls, LE and Rios-Solis, L},
title = {Multiplex Genome Engineering Methods for Yeast Cell Factory Development.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {589468},
pmid = {33195154},
issn = {2296-4185},
abstract = {As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. This is largely due to recent advancements in genome editing methods like CRISPR/Cas tech and high-throughput omics tools. The model organism, baker's yeast (Saccharomyces cerevisiae) is an important synthetic biology chassis for high-value metabolite production. Multiplex genome engineering approaches can expedite the construction and fine tuning of effective heterologous pathways in yeast cell factories. Numerous multiplex genome editing techniques have emerged to capitalize on this recently. This review focuses on recent advancements in such tools, such as delta integration and rDNA cluster integration coupled with CRISPR-Cas tools to greatly enhance multi-integration efficiency. Examples of pre-placed gate systems which are an innovative alternative approach for multi-copy gene integration were also reviewed. In addition to multiple integration studies, multiplexing of alternative genome editing methods are also discussed. Finally, multiplex genome editing studies involving non-conventional yeasts and the importance of automation for efficient cell factory design and construction are considered. Coupling the CRISPR/Cas system with traditional yeast multiplex genome integration or donor DNA delivery methods expedites strain development through increased efficiency and accuracy. Novel approaches such as pre-placing synthetic sequences in the genome along with improved bioinformatics tools and automation technologies have the potential to further streamline the strain development process. In addition, the techniques discussed to engineer S. cerevisiae, can be adapted for use in other industrially important yeast species for cell factory development.},
}
@article {pmid33193271,
year = {2020},
author = {Pickering, AC and Fitzgerald, JR},
title = {The Role of Gram-Positive Surface Proteins in Bacterial Niche- and Host-Specialization.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {594737},
pmid = {33193271},
issn = {1664-302X},
support = {/WT_/Wellcome Trust/United Kingdom ; },
abstract = {Gram-positive bacterial pathogens have an array of proteins on their cell surface that mediate interactions with the host environment. In particular, bacterial cell wall-associated (CWA) proteins play key roles in both colonization and pathogenesis. Furthermore, some CWA proteins promote specialization for host-species or mediate colonization of specific anatomical niches within a host. In this mini review, we provide examples of the many ways by which major pathogens, such as Staphylococci, Streptococci and Listeria monocytogenes, utilize CWA proteins for both host- and niche-specialization. We describe different biological mechanisms mediated by CWA proteins including: the acquisition of iron from hemoglobin in the bloodstream, adherence to and invasion of host cells, and innate immune evasion through binding to the plasma proteins fibrinogen, immunoglobulin G, and complement. We also discuss the limitations of using animal models for understanding the role of specific CWA proteins in host-specialization and how transformative technologies, such as CRISPR-Cas, offer tremendous potential for developing transgenic models that simulate the host environment of interest. Improved understanding of the role of CWA proteins in niche- or host-specificity will allow the design of new therapeutic approaches which target key host-pathogen interactions underpinning Gram-positive bacterial infections.},
}
@article {pmid33193268,
year = {2020},
author = {Cao, Y and Zhou, H and Zhou, X and Li, F},
title = {Control of Plant Viruses by CRISPR/Cas System-Mediated Adaptive Immunity.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {593700},
pmid = {33193268},
issn = {1664-302X},
abstract = {Plant diseases caused by invading plant viruses pose serious threats to agricultural production in the world, and the antiviral engineering initiated by molecular biotechnology has been an effective strategy to prevent and control plant viruses. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-mediated DNA or RNA editing/interference in plants make them very attractive tools applicable to the plant protection field. Here, we review the development of CRISPR/Cas systems and summarize their applications in controlling different plant viruses by targeting viral sequences or host susceptibility genes. We list some potential recessive resistance genes that can be utilized in antiviral breeding and emphasize the importance and promise of recessive resistance gene-based antiviral breeding to generate transgene-free plants without developmental defects. Finally, we discuss the challenges and opportunities for the application of CRISPR/Cas techniques in the prevention and control of plant viruses in the field.},
}
@article {pmid33192264,
year = {2020},
author = {Duarte, F and Déglon, N},
title = {Genome Editing for CNS Disorders.},
journal = {Frontiers in neuroscience},
volume = {14},
number = {},
pages = {579062},
pmid = {33192264},
issn = {1662-4548},
abstract = {Central nervous system (CNS) disorders have a social and economic burden on modern societies, and the development of effective therapies is urgently required. Gene editing may prevent or cure a disease by inducing genetic changes at endogenous loci. Genome editing includes not only the insertion, deletion or replacement of nucleotides, but also the modulation of gene expression and epigenetic editing. Emerging technologies based on ZFs, TALEs, and CRISPR/Cas systems have extended the boundaries of genome manipulation and promoted genome editing approaches to the level of promising strategies for counteracting genetic diseases. The parallel development of efficient delivery systems has also increased our access to the CNS. In this review, we describe the various tools available for genome editing and summarize in vivo preclinical studies of CNS genome editing, whilst considering current limitations and alternative approaches to overcome some bottlenecks.},
}
@article {pmid33191932,
year = {2020},
author = {Loganathan, SK and Malik, A and Langille, E and Luxenburg, C and Schramek, D},
title = {In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {165},
pages = {},
doi = {10.3791/61693},
pmid = {33191932},
issn = {1940-087X},
support = {365252//CIHR/Canada ; },
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Epithelium/metabolism ; *Genetic Testing ; High-Throughput Nucleotide Sequencing ; Integrases/metabolism ; Mice ; Microinjections ; Mouth/*metabolism ; Phenotype ; Skin/*metabolism ; Ultrasonics ; },
abstract = {Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct in vivo CRISPR screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.},
}
@article {pmid33191402,
year = {2020},
author = {Sreedurgalakshmi, K and Srikar, R and Rajkumari, R},
title = {CRISPR-Cas deployment in non-small cell lung cancer for target screening, validations, and discoveries.},
journal = {Cancer gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41417-020-00256-7},
pmid = {33191402},
issn = {1476-5500},
abstract = {Continued advancements in CRISPR-Cas systems have accelerated genome research. Use of CRISPR-Cas in cancer research has been of great interest that is resulting in development of orthogonal methods for drug target validations and discovery of new therapeutic targets through genome-wide screens of cancer cells. CRISPR-based screens have also revealed several new cancer drivers through alterations in tumor suppressor genes (TSGs) and oncogenes inducing resistance to targeted therapies via activation of alternate signaling pathways. Given such dynamic status of cancer, we review the application of CRISPR-Cas in non-small cell lung cancer (NSCLC) for development of mutant models, drug screening, target validation, novel target discoveries, and other emerging potential applications. In addition, CRISPR-based approach for development of novel anticancer combination therapies is also discussed in this review.},
}
@article {pmid33191355,
year = {2020},
author = {Sugihara, H and Kimura, K and Yamanouchi, K and Teramoto, N and Okano, T and Daimon, M and Morita, H and Takenaka, K and Shiga, T and Tanihata, J and Aoki, Y and Inoue-Nagamura, T and Yotsuyanagi, H and Komuro, I},
title = {Age-Dependent Echocardiographic and Pathologic Findings in a Rat Model with Duchenne Muscular Dystrophy Generated by CRISPR/Cas9 Genome Editing.},
journal = {International heart journal},
volume = {61},
number = {6},
pages = {1279-1284},
doi = {10.1536/ihj.20-372},
pmid = {33191355},
issn = {1349-3299},
mesh = {Age Factors ; Animals ; Blood Flow Velocity ; CRISPR-Cas Systems ; Cardiomyopathies/diagnostic imaging/genetics/pathology/*physiopathology ; *Disease Models, Animal ; Dystrophin/*genetics ; Echocardiography ; Frameshift Mutation ; Gene Editing ; Heart/diagnostic imaging/*physiopathology ; Heart Ventricles/diagnostic imaging/pathology/physiopathology ; Male ; Muscular Dystrophy, Duchenne/diagnostic imaging/genetics/pathology/*physiopathology ; Myocardium/*pathology ; *Rats ; },
abstract = {Duchenne muscular dystrophy (DMD) is X-linked recessive myopathy caused by mutations in the dystrophin gene. Although conventional treatments have improved their prognosis, inevitable progressive cardiomyopathy is still the leading cause of death in patients with DMD. To explore novel therapeutic options, a suitable animal model with heart involvement has been warranted.We have generated a rat model with an out-of-frame mutation in the dystrophin gene using CRISPR/Cas9 genome editing (DMD rats). The aim of this study was to evaluate their cardiac functions and pathologies to provide baseline data for future experiments developing treatment options for DMD.In comparison with age-matched wild rats, 6-month-old DMD rats showed no significant differences by echocardiographic evaluations. However, 10-month-old DMD rats showed significant deterioration in left ventricular (LV) fractional shortening (P = 0.024), and in tissue Doppler peak systolic velocity (Sa) at the LV lateral wall (P = 0.041) as well as at the right ventricular (RV) free-wall (P = 0.004). These functional findings were consistent with the fibrotic distributions by histological analysis.Although the cardiac phenotype was milder than anticipated, DMD rats showed similar distributions and progression of heart involvement to those of patients with DMD. This animal may be a useful model with which to develop effective drugs and to understand the underlying mechanisms of progressive heart failure in patients with DMD.},
}
@article {pmid33185863,
year = {2020},
author = {Zhang, Z and Yuan, S and Xu, S and Guo, D and Chen, L and Hou, W and Wang, M},
title = {Suppression of HIV-1 Integration by Targeting HIV-1 Integrase for Degradation with A Chimeric Ubiquitin Ligase.},
journal = {Virologica Sinica},
volume = {},
number = {},
pages = {},
doi = {10.1007/s12250-020-00311-5},
pmid = {33185863},
issn = {1995-820X},
abstract = {Human immunodeficiency virus (HIV) attacks human immune system and causes life-threatening acquired immune deficiency syndrome (AIDS). Treatment with combination antiretroviral therapy (cART) could inhibit virus growth and slow progression of the disease, however, at the same time posing various adverse effects. Host ubiquitin-proteasome pathway (UPP) plays important roles in host immunity against pathogens including viruses by inducing degradation of viral proteins. Previously a series of methods for retargeting substrates for ubiquitin-proteasome degradation have been successfully established. In this study, we attempted to design and construct artificial chimeric ubiquitin ligases (E3s) based on known human E3s in order to manually target HIV-1 integrase for ubiquitin proteasome pathway-mediated degradation. Herein, a series of prototypical chimeric E3s have been designed and constructed, and original substrate-binding domains of these E3s were replaced with host protein domains which interacted with viral proteins. After functional assessment screening, 146LI was identified as a functional chimeric E3 for HIV-1 NL4-3 integrase. 146LI was then further optimized to generate 146LIS (146LI short) which has been shown to induce Lys48-specific polyubiquitination and reduce protein level of HIV-1 NL4-3 integrase more effectively in cells. Lymphocyte cells with 146LIS knock-in generated by CRISPR/Cas-mediated homology-directed repair (HDR) showed remarkably decreased integration of HIV-1 NL4-3 viral DNAs and reduced viral replication without obvious cell cytotoxicity. Our study successfully obtained an artificial chimeric E3 which can induce Lys48-specific polyubiquitination and proteasome-mediated degradation of HIV-1 NL4-3 integrase, thus effectively inhibiting viral DNA integration and viral replication upon virus infection.},
}
@article {pmid33185860,
year = {2020},
author = {Batool, A and Malik, F and Andrabi, KI},
title = {Expansion of the CRISPR/Cas Genome-Sculpting Toolbox: Innovations, Applications and Challenges.},
journal = {Molecular diagnosis &amp; therapy},
volume = {},
number = {},
pages = {},
doi = {10.1007/s40291-020-00500-8},
pmid = {33185860},
issn = {1179-2000},
abstract = {The emergence of the versatile gene-editing technology using programmable sequence-specific endonuclease system (CRISPR-Cas9) has instigated a major upheaval in biomedical research. In a brief span of time, CRISPR/Cas has been adopted by research labs around the globe because of its potential for significant progress and applicability in terms of efficiency, versatility and simplicity. It is a breakthrough technique for systematic genetic engineering, genome labelling, epigenetic and transcriptional modulation, and multiplexed gene editing, amongst others. This review provides an illustrative overview of the current research trends using CRISPR/Cas technology. We highlight the latest developments in CRISPR/Cas technique including CRISPR imaging, discovery of novel CRISPR systems, and applications in altering the genome, epigenome or RNA in different organisms. Finally, we address the potential challenges of this technique for its future use. Development of new CRISPR/Cas systems.},
}
@article {pmid33182769,
year = {2020},
author = {Storey, N and Rabiey, M and Neuman, BW and Jackson, RW and Mulley, G},
title = {Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages.},
journal = {Viruses},
volume = {12},
number = {11},
pages = {},
pmid = {33182769},
issn = {1999-4915},
abstract = {Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonastolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonasagarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.},
}
@article {pmid33180479,
year = {2020},
author = {Greisch, JF and van der Laarse, SAM and Heck, AJR},
title = {Enhancing Top-Down Analysis Using Chromophore-Assisted Infrared Multiphoton Dissociation from (Phospho)peptides to Protein Assemblies.},
journal = {Analytical chemistry},
volume = {92},
number = {23},
pages = {15506-15516},
pmid = {33180479},
issn = {1520-6882},
abstract = {Infrared multiphoton dissociation (IRMPD) has been used in mass spectrometry to fragment peptides and proteins, providing fragments mostly similar to collisional activation. Using the 10.6 μm wavelength of a CO2 laser, IRMPD suffers from the relative low absorption cross-section of peptides and small proteins. Focusing on top-down analysis, we investigate different means to tackle this issue. We first reassess efficient sorting of phosphopeptides from nonphosphopeptides based on IR-absorption cross-sectional enhancement by phosphate moieties. We subsequently demonstrate that a myo-inositol hexakisphosphate (IP6) noncovalent adduct can substantially enhance IRMPD for nonphosphopeptides and that this strategy can be extended to proteins. As a natural next step, we show that native phospho-proteoforms of proteins display a distinct and enhanced fragmentation, compared to their unmodified counterparts, facilitating phospho-group site localization. We then evaluate the impact of size on the IRMPD of proteins and their complexes. When applied to protein complexes ranging from a 365 kDa CRISPR-Cas Csy ribonucleoprotein hetero-decamer, a 800 kDa GroEL homo-tetradecamer in its apo-form or loaded with its ATP cofactor, to a 1 MDa capsid-like homo-hexacontamer, we conclude that while phosphate moieties present in crRNA and ATP molecules enhance IRMPD, an increase in the IR cross-section with the size of the protein assembly also favorably accrues dissociation yields. Overall, our work showcases the versatility of IRMPD in the top-down analysis of peptides, phosphopeptides, proteins, phosphoproteins, ribonucleoprotein assemblies, and large protein complexes.},
}
@article {pmid33180295,
year = {2021},
author = {Hwang, GH and Bae, S},
title = {Web-Based Base Editing Toolkits: BE-Designer and BE-Analyzer.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2189},
number = {},
pages = {81-88},
doi = {10.1007/978-1-0716-0822-7_7},
pmid = {33180295},
issn = {1940-6029},
abstract = {The CRISPR-Cas system is broadly used for genome editing because of its convenience and relatively low cost. However, the use of CRISPR nucleases to induce specific nucleotide changes in target DNA requires complex procedures and additional donor DNAs. Furthermore, CRISPR nuclease-mediated DNA cleavage at target sites frequently causes large deletions or genomic rearrangements. In contrast, base editors that consist of catalytically dead Cas9 (dCas9) or Cas9 nickase (nCas9) connected to a cytidine or a guanine deaminase can correct point mutations in the absence of additional donor DNA and without generating double-strand breaks (DSBs) in the target region. To design target sites and assess mutation ratios for cytosine and adenine base editors (CBEs and ABEs), we have developed web tools, named BE-Designer and BE-Analyzer. These tools are easy to use (such that tasks are accomplished by clicking on relevant buttons) and do not require a deep knowledge of bioinformatics.},
}
@article {pmid33179250,
year = {2020},
author = {Benler, S and Koonin, EV},
title = {Phage lysis-lysogeny switches and programmed cell death: Danse macabre.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {42},
number = {12},
pages = {e2000114},
doi = {10.1002/bies.202000114},
pmid = {33179250},
issn = {1521-1878},
abstract = {Exploration of immune systems in prokaryotes, such as restriction-modification or CRISPR-Cas, shows that both innate and adaptive systems possess programmed cell death (PCD) potential. The key outstanding question is how the immune systems sense and &quot;predict&quot; infection outcomes to &quot;decide&quot; whether to fight the pathogen or induce PCD. There is a striking parallel between this life-or-death decision faced by the cell and the decision by temperate viruses to protect or kill their hosts, epitomized by the lysis-lysogeny switch of bacteriophage Lambda. Immune systems and temperate phages sense the same molecular inputs, primarily, DNA damage, that determine whether the cell lives or dies. Because temperate (pro)phages are themselves components of prokaryotic genomes, their shared &quot;interests&quot; with the hosts result in coregulation of the lysis-lysogeny switch and immune systems that jointly provide the cell with the decision machinery to probe and predict infection outcomes, answering the life-or-death question.},
}
@article {pmid33178158,
year = {2020},
author = {Mancilla-Rojano, J and Ochoa, SA and Reyes-Grajeda, JP and Flores, V and Medina-Contreras, O and Espinosa-Mazariego, K and Parra-Ortega, I and Rosa-Zamboni, D and Castellanos-Cruz, MDC and Arellano-Galindo, J and Cevallos, MA and Hernández-Castro, R and Xicohtencatl-Cortes, J and Cruz-Córdova, A},
title = {Molecular Epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii Complex Isolated From Children at the Hospital Infantil de México Federico Gómez.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {576673},
pmid = {33178158},
issn = {1664-302X},
abstract = {The Acinetobacter calcoaceticus-baumannii (Acb) complex is regarded as a group of phenotypically indistinguishable opportunistic pathogens responsible for mainly causing hospital-acquired pneumonia and bacteremia. The aim of this study was to determine the frequency of isolation of the species that constitute the Acb complex, as well as their susceptibility to antibiotics, and their distribution at the Hospital Infantil de Mexico Federico Gomez (HIMFG). A total of 88 strains previously identified by Vitek 2®, 40 as Acinetobacter baumannii and 48 as Acb complex were isolated from 52 children from 07, January 2015 to 28, September 2017. A. baumannii accounted for 89.77% (79/88) of the strains; Acinetobacter pittii, 6.82% (6/88); and Acinetobacter nosocomialis, 3.40% (3/88). Most strains were recovered mainly from patients in the intensive care unit (ICU) and emergency wards. Blood cultures (BC) provided 44.32% (39/88) of strains. The 13.63% (12/88) of strains were associated with primary bacteremia, 3.4% (3/88) with secondary bacteremia, and 2.3% (2/88) with pneumonia. In addition, 44.32% (39/88) were multidrug-resistant (MDR) strains and, 11.36% (10/88) were extensively drug-resistant (XDR). All strains amplified the blaOXA-51 gene; 51.13% (45/88), the blaOXA-23 gene; 4.54% (4/88), the blaOXA-24 gene; and 2.27% (2/88), the blaOXA-58 gene. Plasmid profiles showed that the strains had 1-6 plasmids. The strains were distributed in 52 pulsotypes, and 24 showed identical restriction patterns, with a correlation coefficient of 1.0. Notably, some strains with the same pulsotype were isolated from different patients, wards, or years, suggesting the persistence of more than one clone. Twenty-seven sequence types (STs) were determined for the strains based on a Pasteur multilocus sequence typing (MLST) scheme using massive sequencing; the most prevalent was ST 156 (27.27%, 24/88). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas I-Fb system provided amplification in A. baumannii and A. pittii strains (22.73%, 20/88). This study identified an increased number of MDR strains and the relationship among strains through molecular typing. The data suggest that more than one strain could be causing an infection in some patient. The implementation of molecular epidemiology allowed the characterization of a set of strains and identification of different attributes associated with its distribution in a specific environment.},
}
@article {pmid33177521,
year = {2020},
author = {Du, K and Luo, Q and Yin, L and Wu, J and Liu, Y and Gan, J and Dong, A and Shen, WH},
title = {OsChz1 acts as a histone chaperone in modulating chromatin organization and genome function in rice.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5717},
pmid = {33177521},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Chromatin/genetics/*metabolism ; DNA Methylation ; Flowers/metabolism ; Gene Expression Regulation, Plant ; Genome, Plant ; Histones/genetics/*metabolism ; Molecular Chaperones/genetics/metabolism ; Mutation ; Nucleosomes/genetics ; Oryza/*genetics/growth &amp; development/metabolism ; Phylogeny ; Plant Proteins/genetics/*metabolism ; Plants, Genetically Modified ; Protein Multimerization ; },
abstract = {While the yeast Chz1 acts as a specific histone-chaperone for H2A.Z, functions of CHZ-domain proteins in multicellular eukaryotes remain obscure. Here, we report on the functional characterization of OsChz1, a sole CHZ-domain protein identified in rice. OsChz1 interacts with both the canonical H2A-H2B dimer and the variant H2A.Z-H2B dimer. Within crystal structure the C-terminal region of OsChz1 binds H2A-H2B via an acidic region, pointing to a previously unknown recognition mechanism. Knockout of OsChz1 leads to multiple plant developmental defects. At genome-wide level, loss of OsChz1 causes mis-regulations of thousands of genes and broad alterations of nucleosome occupancy as well as reductions of H2A.Z-enrichment. While OsChz1 associates with chromatin regions enriched of repressive histone marks (H3K27me3 and H3K4me2), its loss does not affect the genome landscape of DNA methylation. Taken together, it is emerging that OsChz1 functions as an important H2A/H2A.Z-H2B chaperone in dynamic regulation of chromatin for higher eukaryote development.},
}
@article {pmid33175893,
year = {2020},
author = {Smirnikhina, SA and Kondrateva, EV and Adilgereeva, EP and Anuchina, AA and Zaynitdinova, MI and Slesarenko, YS and Ershova, AS and Ustinov, KD and Yasinovsky, MI and Amelina, EL and Voronina, ES and Yakushina, VD and Tabakov, VY and Lavrov, AV},
title = {P.F508del editing in cells from cystic fibrosis patients.},
journal = {PloS one},
volume = {15},
number = {11},
pages = {e0242094},
pmid = {33175893},
issn = {1932-6203},
abstract = {Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out.},
}
@article {pmid33174655,
year = {2020},
author = {Ye, Q and Sung, TC and Yang, JM and Ling, QD and He, Y and Higuchi, A},
title = {Generation of universal and hypoimmunogenic human pluripotent stem cells.},
journal = {Cell proliferation},
volume = {53},
number = {12},
pages = {e12946},
pmid = {33174655},
issn = {1365-2184},
support = {108-2221-E-008-037//Ministry of Science and Technology, Taiwan/ ; 108-2221-E-008-057//Ministry of Science and Technology, Taiwan/ ; 81701032//National Natural Science Foundation of China/ ; 81871503//National Natural Science Foundation of China/ ; 18K05251//Japan Society for the Promotion of Science/ ; },
mesh = {CRISPR-Cas Systems/*genetics ; Family Characteristics ; *Gene Editing ; Humans ; Killer Cells, Natural/*cytology ; Pluripotent Stem Cells/*cytology ; Stem Cell Transplantation/methods ; },
abstract = {There is a need to store very large numbers of conventional human pluripotent stem cell (hPSC) lines for their off-the-shelf usage in stem cell therapy. Therefore, it is valuable to generate &quot;universal&quot; or &quot;hypoimmunogenic&quot; hPSCs with gene-editing technology by knocking out or in immune-related genes. A few universal or hypoimmunogenic hPSC lines should be enough to store for their off-the-shelf usage. Here, we overview and discuss how to prepare universal or hypoimmunogenic hPSCs and their disadvantages. β2-Microglobulin-knockout hPSCs did not harbour human leukocyte antigen (HLA)-expressing class I cells but rather activated natural killer (NK) cells. To avoid NK cell and macrophage activities, homozygous hPSCs expressing a single allele of an HLA class I molecule, such as HLA-C, were developed. Major HLA class I molecules were knocked out, and PD-L1, HLA-G and CD47 were knocked in hPSCs using CRISPR/Cas9 gene editing. These cells escaped activation of not only T cells but also NK cells and macrophages, generating universal hPSCs.},
}
@article {pmid33172134,
year = {2020},
author = {Zink, IA and Wimmer, E and Schleper, C},
title = {Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales.},
journal = {Biomolecules},
volume = {10},
number = {11},
pages = {},
pmid = {33172134},
issn = {2218-273X},
support = {695192/ERC_/European Research Council/International ; P29399//Austrian Science Fund/ ; DOC-fellowship//Österreichischen Akademie der Wissenschaften/ ; DOC completion grant//Universität Wien/ ; },
abstract = {Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.},
}
@article {pmid33171122,
year = {2020},
author = {Lahey, LJ and Mardjuki, RE and Wen, X and Hess, GT and Ritchie, C and Carozza, JA and Böhnert, V and Maduke, M and Bassik, MC and Li, L},
title = {LRRC8A:C/E Heteromeric Channels Are Ubiquitous Transporters of cGAMP.},
journal = {Molecular cell},
volume = {80},
number = {4},
pages = {578-591.e5},
doi = {10.1016/j.molcel.2020.10.021},
pmid = {33171122},
issn = {1097-4164},
support = {DP2 CA228044/CA/NCI NIH HHS/United States ; DP2 HD084069/HD/NICHD NIH HHS/United States ; },
mesh = {Biological Transport ; *CRISPR-Cas Systems ; Cyclopentanes/pharmacology ; Humans ; Indans/pharmacology ; Lysophospholipids/pharmacology ; Membrane Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Nucleotides, Cyclic/*metabolism ; Sphingosine/analogs &amp; derivatives/pharmacology ; U937 Cells ; },
abstract = {Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.},
}
@article {pmid33170255,
year = {2020},
author = {Bennett, EP and Petersen, BL and Johansen, IE and Niu, Y and Yang, Z and Chamberlain, CA and Met, Ö and Wandall, HH and Frödin, M},
title = {INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {11958-11981},
doi = {10.1093/nar/gkaa975},
pmid = {33170255},
issn = {1362-4962},
mesh = {Animals ; *CRISPR-Cas Systems ; Cloning, Organism/methods ; DNA/*genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Repair ; Gene Editing/*methods ; Gene Knockout Techniques ; *Genome ; Humans ; *INDEL Mutation ; Mice ; Sheep/genetics ; Solanum tuberosum/genetics ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Zinc Finger Nucleases/genetics/metabolism ; },
abstract = {Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.},
}
@article {pmid33167031,
year = {2020},
author = {Galperin, MY and Wolf, YI and Makarova, KS and Vera Alvarez, R and Landsman, D and Koonin, EV},
title = {COG database update: focus on microbial diversity, model organisms, and widespread pathogens.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa1018},
pmid = {33167031},
issn = {1362-4962},
abstract = {The Clusters of Orthologous Genes (COG) database, also referred to as the Clusters of Orthologous Groups of proteins, was created in 1997 and went through several rounds of updates, most recently, in 2014. The current update, available at https://www.ncbi.nlm.nih.gov/research/COG, substantially expands the scope of the database to include complete genomes of 1187 bacteria and 122 archaea, typically, with a single genome per genus. In addition, the current version of the COGs includes the following new features: (i) the recently deprecated NCBI's gene index (gi) numbers for the encoded proteins are replaced with stable RefSeq or GenBank\ENA\DDBJ coding sequence (CDS) accession numbers; (ii) COG annotations are updated for >200 newly characterized protein families with corresponding references and PDB links, where available; (iii) lists of COGs grouped by pathways and functional systems are added; (iv) 266 new COGs for proteins involved in CRISPR-Cas immunity, sporulation in Firmicutes and photosynthesis in cyanobacteria are included; and (v) the database is made available as a web page, in addition to FTP. The current release includes 4877 COGs. Future plans include further expansion of the COG collection by adding archaeal COGs (arCOGs), splitting the COGs containing multiple paralogs, and continued refinement of COG annotations.},
}
@article {pmid33166385,
year = {2020},
author = {Guo, R and Xu, Y and Leu, NA and Zhang, L and Fuchs, SY and Ye, L and Wang, PJ},
title = {The ssDNA-binding protein MEIOB acts as a dosage-sensitive regulator of meiotic recombination.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12219-12233},
doi = {10.1093/nar/gkaa1016},
pmid = {33166385},
issn = {1362-4962},
support = {R35 GM118052/GM/NIGMS NIH HHS/United States ; },
abstract = {Meiotic recombination enables reciprocal exchange of genetic information between parental chromosomes and is essential for fertility. MEIOB, a meiosis-specific ssDNA-binding protein, regulates early meiotic recombination. Here we report that the human infertility-associated missense mutation (N64I) in MEIOB causes protein degradation and reduced crossover formation in mouse testes. Although the MEIOB N64I substitution is associated with human infertility, the point mutant mice are fertile despite meiotic defects. Meiob mutagenesis identifies serine 67 as a critical residue for MEIOB. Biochemically, these two mutations (N64I and S67 deletion) cause self-aggregation of MEIOB and sharply reduced protein half-life. Molecular genetic analyses of both point mutants reveal an important role for MEIOB in crossover formation in late meiotic recombination. Furthermore, we find that the MEIOB protein levels directly correlate with the severity of meiotic defects. Our results demonstrate that MEIOB regulates meiotic recombination in a dosage-dependent manner.},
}
@article {pmid33166100,
year = {2020},
author = {Becker, A and Mannebach, S and Mathar, I and Weissgerber, P and Freichel, M and Loodin, AP and Fecher-Trost, C and Belkacemi, A and Beck, A and Philipp, SE},
title = {Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {54},
number = {6},
pages = {1115-1131},
doi = {10.33594/000000304},
pmid = {33166100},
issn = {1421-9778},
support = {CRC 894 A3, CRC 894 A14, CRC 894 P2, CRC 1188 B02//DFG (Deutsche Forschungsgemeinschaft)/Germany ; },
abstract = {BACKGROUND/AIMS: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca2+ influx into pancreatic beta cells by the opening of voltage-gated Ca2+ channels. Transient Receptor Potential Melastatin 3 proteins build Ca2+ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca2+ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release.<br><br>METHODS: We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca2+ channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests.<br><br>RESULTS: TRPM3 channels triggered the activity of voltage-gated Ca2+ channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca2+ signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca2+ signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose.<br><br>CONCLUSION: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release.},
}
@article {pmid33159392,
year = {2021},
author = {Li, H and Cui, X and Sun, L and Deng, X and Liu, S and Zou, X and Li, B and Wang, C and Wang, Y and Liu, Y and Lu, B and Cao, B},
title = {High concentration of Cas12a effector tolerates more mismatches on ssDNA.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {35},
number = {1},
pages = {e21153},
doi = {10.1096/fj.202001475R},
pmid = {33159392},
issn = {1530-6860},
abstract = {Rapid pathogen detection is critical for prompt treatment, interrupting transmission routes, and decreasing morbidity and mortality. The V-type CRISPR system had been used for rapid pathogen detection. However, whether single-stranded DNA in CRISPR system can cause false positives remains undetermined. Herein, we show that high molar concentration of Cas12a effector tolerated more mismatches on ssDNA and activated its trans-cleavage activity at six base matches. Reducing Cas12a and crRNA molar concentration increased the minimal base-match number required for Cas12a ssDNA activation to 11, which reducing nonspecific activation. We then established a Cas12a-based M tuberculosis detection system with a primer having an 8 bp overlap with crRNA. This system did not exhibit primer-induced false positives, and minimum detection copy reached 1 copy/uL (inputting 1-μL sample) in standard strains. The Cas12a-based M tuberculosis detection system showed 80.0% sensitivity and 100.0% specificity in verification using clinical specimens, compared with Xpert MTB/RIF, which showed 72.0% sensitivity and 90.9% specificity. All these results prove that appropriate concentration of cas12a effector can effectively perform nucleic acid detection.},
}
@article {pmid33159140,
year = {2020},
author = {Osakabe, K and Wada, N and Miyaji, T and Murakami, E and Marui, K and Ueta, R and Hashimoto, R and Abe-Hara, C and Kong, B and Yano, K and Osakabe, Y},
title = {Genome editing in plants using CRISPR type I-D nuclease.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {648},
pmid = {33159140},
issn = {2399-3642},
abstract = {Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I-the most abundant CRISPR system in bacteria-has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.},
}
@article {pmid33159058,
year = {2020},
author = {Pinilla-Redondo, R and Shehreen, S and Marino, ND and Fagerlund, RD and Brown, CM and Sørensen, SJ and Fineran, PC and Bondy-Denomy, J},
title = {Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5652},
pmid = {33159058},
issn = {2041-1723},
support = {F32 GM133127/GM/NIGMS NIH HHS/United States ; DP5 OD021344/OD/NIH HHS/United States ; R01 GM127489/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacteria/*genetics/immunology/virology ; Bacterial Proteins/genetics/*immunology ; Bacteriophages/genetics/physiology ; *CRISPR-Cas Systems ; Interspersed Repetitive Sequences ; Multigene Family ; },
abstract = {Many prokaryotes employ CRISPR-Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have developed strategies to bypass immunity, including anti-CRISPR (Acr) proteins; yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we report the discovery of new Acrs by assaying candidate genes adjacent to a conserved Acr-associated (Aca) gene, aca5, against a panel of six type I systems: I-F (Pseudomonas, Pectobacterium, and Serratia), I-E (Pseudomonas and Serratia), and I-C (Pseudomonas). We uncover 11 type I-F and/or I-E anti-CRISPR genes encoded on chromosomal and extrachromosomal MGEs within Enterobacteriaceae and Pseudomonas, and an additional Aca (aca9). The acr genes not only associate with other acr genes, but also with genes encoding inhibitors of distinct bacterial defense systems. Thus, our findings highlight the potential exploitation of acr loci neighborhoods for the identification of previously undescribed anti-defense systems.},
}
@article {pmid33157047,
year = {2020},
author = {Hendriks, D and Clevers, H and Artegiani, B},
title = {CRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids.},
journal = {Cell stem cell},
volume = {27},
number = {5},
pages = {705-731},
doi = {10.1016/j.stem.2020.10.014},
pmid = {33157047},
issn = {1875-9777},
abstract = {CRISPR-Cas technology has revolutionized biological research and holds great therapeutic potential. Here, we review CRISPR-Cas systems and their latest developments with an emphasis on application to human cells. We also discuss how different CRISPR-based strategies can be used to accomplish a particular genome engineering goal. We then review how different CRISPR tools have been used in genome engineering of human stem cells in vitro, covering both the pluripotent (iPSC/ESC) and somatic adult stem cell fields and, in particular, 3D organoid cultures. Finally, we discuss the progress and challenges associated with CRISPR-based genome editing of human stem cells for therapeutic use.},
}
@article {pmid33155277,
year = {2020},
author = {Murakami, Y and Futamata, R and Horibe, T and Ueda, K and Kinoshita, M},
title = {CRISPR/Cas9 nickase-mediated efficient and seamless knock-in of lethal genes in the medaka fish Oryzias latipes.},
journal = {Development, growth &amp; differentiation},
volume = {62},
number = {9},
pages = {554-567},
doi = {10.1111/dgd.12700},
pmid = {33155277},
issn = {1440-169X},
support = {17H03860//Grant-in-Aid for Scientific Research (B)/ ; 18J10121//Grant-in-Aid for JSPS fellows/ ; 18H05269//Grant-in-Aid for Scientific Research (S)/ ; },
abstract = {The CRISPR/Cas system offers new opportunities for targeted gene modifications in a wide range of organisms. In medaka (Oryzias latipes), a vertebrate model organism, a wild-type Cas9-based approach is commonly used to establish desired strains, however, its use in lethal genes is still challenging due to excess gene disruptions triggered by DNA double strand breaks (DSBs). To overcome this problem, we aimed to develop a new knock-in system using Cas9 nickase (Cas9n) that can reduce DNA DSBs. We revealed that Cas9n allowed reduction of the DSB-induced unwanted mutagenesis via non-homologous end-joining at both on- and off- target sites. Further, with a new donor plasmid (p2BaitD) that provides a linear template through Cas9n-mediated nicks, we successfully integrated reporter cassettes via homology-directed repair (HDR) into all three loci tested, including a lethal gene. In the experiment targeting the lethal gene, the combination of p2BaitD and Cas9n achieved higher survival rates than the Cas9-based approach, which enabled the desired knock-in founders. Additionally, through a technical blend of our knock-in system with a recently developed One-step mating protocol, we successfully established a homozygous knock-in strain in one generation period. This study presents evidence of an effective method to generate an HDR-mediated gene knock-in in medaka and other organisms, which is useful for establishing screening platforms for genes or drugs toxicity or other applications.},
}
@article {pmid33154469,
year = {2020},
author = {Oo, JA and Irmer, B and Günther, S and Warwick, T and Pálfi, K and Izquierdo Ponce, J and Teichmann, T and Pflüger-Müller, B and Gilsbach, R and Brandes, RP and Leisegang, MS},
title = {ZNF354C is a transcriptional repressor that inhibits endothelial angiogenic sprouting.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {19079},
pmid = {33154469},
issn = {2045-2322},
abstract = {Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We recently discovered that endothelial cells harbour a specific mechanism to limit the action of ZNF354C, whose function in endothelial cells is unknown. Given that ZNF354C has so far only been studied in bone and tumour, its function was determined in endothelial cells. ZNF354C is expressed in vascular cells and localises to the nucleus and cytoplasm. Overexpression of ZNF354C in human endothelial cells results in a marked inhibition of endothelial sprouting. RNA-sequencing of human microvascular endothelial cells with and without overexpression of ZNF354C revealed that the protein is a potent transcriptional repressor. ZNF354C contains an active KRAB domain which mediates this suppression as shown by mutagenesis analysis. ZNF354C interacts with dsDNA, TRIM28 and histones, as observed by proximity ligation and immunoprecipitation. Moreover, chromatin immunoprecipitation revealed that the ZNF binds to specific endothelial-relevant target-gene promoters. ZNF354C suppresses these genes as shown by CRISPR/Cas knockout and RNAi. Inhibition of endothelial sprouting by ZNF354C is dependent on the amino acids DV and MLE of the KRAB domain. These results demonstrate that ZNF354C is a repressive transcription factor which acts through a KRAB domain to inhibit endothelial angiogenic sprouting.},
}
@article {pmid33153299,
year = {2020},
author = {Mayuramart, O and Nimsamer, P and Rattanaburi, S and Chantaravisoot, N and Khongnomnan, K and Chansaenroj, J and Puenpa, J and Suntronwong, N and Vichaiwattana, P and Poovorawan, Y and Payungporn, S},
title = {Detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses based on CRISPR-Cas12a.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {},
number = {},
pages = {1535370220963793},
doi = {10.1177/1535370220963793},
pmid = {33153299},
issn = {1535-3699},
abstract = {Due to the common symptoms of COVID-19, patients are similar to influenza-like illness. Therefore, the detection method would be crucial to discriminate between SARS-CoV-2 and influenza virus-infected patients. In this study, CRISPR-Cas12a-based detection was applied for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus which would be a practical and attractive application for screening of patients with COVID-19 and influenza in areas with limited resources. The limit of detection for SARS-CoV-2, influenza A, and influenza B detection was 10, 103, and 103 copies/reaction, respectively. Moreover, the assays yielded no cross-reactivity against other respiratory viruses. The results revealed that the detection of influenza virus and SARS-CoV-2 by using RT-RPA and CRISPR-Cas12a technology reaches 96.23% sensitivity and 100% specificity for SARS-CoV-2 detection. The sensitivity for influenza virus A and B detections was 85.07% and 94.87%, respectively. In addition, the specificity for influenza virus A and B detections was approximately 96%. In conclusion, the RT-RPA with CRISPR-Cas12a assay was an effective method for the screening of influenza viruses and SARS-CoV-2 which could be applied to detect other infectious diseases in the future.},
}
@article {pmid33152994,
year = {2020},
author = {Lugli, GA and Tarracchini, C and Alessandri, G and Milani, C and Mancabelli, L and Turroni, F and Neuzil-Bunesova, V and Ruiz, L and Margolles, A and Ventura, M},
title = {Decoding the Genomic Variability among Members of the Bifidobacteriumdentium Species.},
journal = {Microorganisms},
volume = {8},
number = {11},
pages = {},
pmid = {33152994},
issn = {2076-2607},
abstract = {Members of the Bifidobacterium dentium species are usually identified in the oral cavity of humans and associated with the development of plaque and dental caries. Nevertheless, they have also been detected from fecal samples, highlighting a widespread distribution among mammals. To explore the genetic variability of this species, we isolated and sequenced the genomes of 18 different B. dentium strains collected from fecal samples of several primate species and an Ursus arctos. Thus, we investigated the genomic variability and metabolic abilities of the new B. dentium isolates together with 20 public genome sequences. Comparative genomic analyses provided insights into the vast metabolic repertoire of the species, highlighting 19 glycosyl hydrolases families shared between each analyzed strain. Phylogenetic analysis of the B. dentium taxon, involving 1140 conserved genes, revealed a very close phylogenetic relatedness among members of this species. Furthermore, low genomic variability between strains was also confirmed by an average nucleotide identity analysis showing values higher than 98.2%. Investigating the genetic features of each strain, few putative functional mobile elements were identified. Besides, a consistent occurrence of defense mechanisms such as CRISPR-Cas and restriction-modification systems may be responsible for the high genome synteny identified among members of this taxon.},
}
@article {pmid33152914,
year = {2020},
author = {Lotfi, M and Rezaei, N},
title = {CRISPR/Cas13: A potential therapeutic option of COVID-19.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {131},
number = {},
pages = {110738},
doi = {10.1016/j.biopha.2020.110738},
pmid = {33152914},
issn = {1950-6007},
mesh = {Betacoronavirus/drug effects/*genetics ; COVID-19 ; CRISPR-Associated Proteins/pharmacology ; *CRISPR-Cas Systems ; Conserved Sequence ; Coronavirus Infections/drug therapy/genetics/*therapy ; *Gene Editing ; Genetic Therapy/*methods ; Genome, Viral ; Humans ; Pandemics ; Pneumonia, Viral/genetics/*therapy ; RNA, Guide/genetics ; RNA, Viral/antagonists &amp; inhibitors/*genetics ; SARS-CoV-2 ; },
abstract = {The novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be considered as the most important current global issue, as it has caused the novel coronavirus disease (COVID-19) pandemic, which has resulted in high mortality and morbidity rates all around the world. Although scientists are trying to discover novel therapies and develop and evaluate various previous treatments, at the time of writing this paper, there was no definite therapy and vaccine for COVID-19. So, as COVID-19 has called ideas for treatment, controlling, and diagnosis, we discussed the application of Clustered Regularly Interspaced Short Palindromic Repeats/Cas13 (CRISPR/Cas13) as a treatment of COVID-19, which received less attention compared with other potential therapeutic options.},
}
@article {pmid33152605,
year = {2020},
author = {Fenster, JA and Eckert, CA},
title = {High-Throughput Functional Genomics for Energy Production.},
journal = {Current opinion in biotechnology},
volume = {67},
number = {},
pages = {7-14},
doi = {10.1016/j.copbio.2020.09.010},
pmid = {33152605},
issn = {1879-0429},
abstract = {Functional genomics remains a foundational field for establishing genotype-phenotype relationships that enable strain engineering. High-throughput (HTP) methods accelerate the Design-Build-Test-Learn cycle that currently drives synthetic biology towards a forward engineering future. Trackable mutagenesis techniques including transposon insertion sequencing and CRISPR-Cas-mediated genome editing allow for rapid fitness profiling of a collection, or library, of mutants to discover beneficial mutations. Due to the relative speed of these experiments compared to adaptive evolution experiments, iterative rounds of mutagenesis can be implemented for next-generation metabolic engineering efforts to design complex production and tolerance phenotypes. Additionally, the expansion of these mutagenesis techniques to novel bacteria are opening up industrial microbes that show promise for establishing a bio-based economy.},
}
@article {pmid33152077,
year = {2020},
author = {Fedorova, I and Vasileva, A and Selkova, P and Abramova, M and Arseniev, A and Pobegalov, G and Kazalov, M and Musharova, O and Goryanin, I and Artamonova, D and Zyubko, T and Shmakov, S and Artamonova, T and Khodorkovskii, M and Severinov, K},
title = {PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {12297-12309},
doi = {10.1093/nar/gkaa998},
pmid = {33152077},
issn = {1362-4962},
abstract = {CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.},
}
@article {pmid33149284,
year = {2020},
author = {Zhu, H and Li, C and Gao, C},
title = {Author Correction: Applications of CRISPR-Cas in agriculture and plant biotechnology.},
journal = {Nature reviews. Molecular cell biology},
volume = {21},
number = {12},
pages = {782},
doi = {10.1038/s41580-020-00312-y},
pmid = {33149284},
issn = {1471-0080},
abstract = {An amendment to this paper has been published and can be accessed via a link at the top of the paper.},
}
@article {pmid33148808,
year = {2020},
author = {Ramachandran, A and Huyke, DA and Sharma, E and Sahoo, MK and Huang, C and Banaei, N and Pinsky, BA and Santiago, JG},
title = {Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {47},
pages = {29518-29525},
pmid = {33148808},
issn = {1091-6490},
mesh = {COVID-19 Nucleic Acid Testing/*methods ; *CRISPR-Cas Systems ; Humans ; Isotachophoresis/*methods ; Microfluidics/*methods ; Nasal Mucosa/virology ; SARS-CoV-2/genetics/isolation &amp; purification ; },
abstract = {The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.},
}
@article {pmid33148705,
year = {2020},
author = {Mustafa, MI and Makhawi, AM},
title = {SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases.},
journal = {Journal of clinical microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1128/JCM.00745-20},
pmid = {33148705},
issn = {1098-660X},
abstract = {Infectious diseases are one of the most intimidating threats to human race, responsible for an immense burden of disabilities and deaths. Rapid diagnosis and treatment of infectious diseases is a better understanding of its pathogenesis. According to WHO, the ideal approach for detecting foreign pathogens should be rapid, specific, sensitive, instrument-free and cost-effective. Nucleic acid pathogen detection methods, typically PCR have numerous limitations, such as highly sophisticated equipments, reagents, and trained personnel rely on well-established laboratories beside time-consuming. Thus, there is a crucial need to develop novel nucleic acid detection tools with rapid, specific, sensitive, and cost-effective, particularly ones that can be used for versatile point-of-care diagnostic applications. Two new methods exploit on unpredicted in vitro properties CRISPR-Cas effectors, turning activated nucleases into basic amplifiers of a specific nucleic-acid binding event. These effectors are attached with a diversity of reporters and utilized in tandem with present of isothermal amplification approaches to create sensitive identification in multiple field deployable formats. Although still in their beginning, yet SHERLOCK and DETECTR technologies are potential methods for rapid detection and identification of infectious disease, with ultra-sensitive tests that don't require a lot of complicated processing. This review described SHERLOCK and DETECTR technologies beside their properties, functions, and perspectives to become the ultimate diagnostic tools for diagnosing infectious diseases and curbing disease outbreaks.},
}
@article {pmid33147453,
year = {2020},
author = {Marina, RJ and Brannan, KW and Dong, KD and Yee, BA and Yeo, GW},
title = {Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing.},
journal = {Cell reports},
volume = {33},
number = {5},
pages = {108350},
doi = {10.1016/j.celrep.2020.108350},
pmid = {33147453},
issn = {2211-1247},
support = {R01 EY029166/EY/NEI NIH HHS/United States ; F31 NS111859/NS/NINDS NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; K22 NS112678/NS/NINDS NIH HHS/United States ; R01 NS103172/NS/NINDS NIH HHS/United States ; },
abstract = {Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3' modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3' gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.},
}
@article {pmid33144569,
year = {2020},
author = {van den Boomen, DJH and Sienkiewicz, A and Berlin, I and Jongsma, MLM and van Elsland, DM and Luzio, JP and Neefjes, JJC and Lehner, PJ},
title = {A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5559},
pmid = {33144569},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; 100140/WT_/Wellcome Trust/United Kingdom ; MR/R0009015/1/MRC_/Medical Research Council/United Kingdom ; 084957/Z/08/Z/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Biological Transport ; CRISPR-Cas Systems/genetics ; Cholesterol/*metabolism ; Cholesterol, LDL/metabolism ; Endosomes/metabolism/ultrastructure ; Fluorescent Dyes/metabolism ; Genome, Human ; Guanine Nucleotide Exchange Factors/metabolism ; HEK293 Cells ; HeLa Cells ; Homeostasis ; Humans ; Hydroxymethylglutaryl-CoA Synthase/metabolism ; Intracellular Signaling Peptides and Proteins/*metabolism ; Lysosomes/*metabolism/ultrastructure ; Models, Biological ; Multiprotein Complexes/metabolism ; Protein Binding ; *Protein Multimerization ; rab GTP-Binding Proteins/*metabolism ; },
abstract = {Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.},
}
@article {pmid33140167,
year = {2020},
author = {Yu, Z and Jiang, S and Wang, Y and Tian, X and Zhao, P and Xu, J and Feng, M and She, Q},
title = {CRISPR-Cas adaptive immune systems in Sulfolobales: genetic studies and molecular mechanisms.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
doi = {10.1007/s11427-020-1745-0},
pmid = {33140167},
issn = {1869-1889},
abstract = {CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria. Organisms of Sulfolobales, an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum, usually contain both type I and type III CRISPR-Cas systems. Two species, Saccharolobus solfataricus and Sulfolobus islandicus, have been important models for CRISPR study in archaea, and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps: adaptation, expression and crRNA processing, and interference. Four subtypes of CRISPR-Cas systems are common in these organisms, including I-A, I-D, III-B, and III-D. These cas genes form functional modules, e.g., all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and iCas modules. Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea, and these methodologies are useful in demonstration of the protospacer-adjacent motif (PAM)-dependent DNA interference by I-A interference modules and multiple interference activities by III-B Cmr systems. Ribonucleoprotein effector complexes have been isolated for Sulfolobales III-B and III-D systems, and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.},
}
@article {pmid33139946,
year = {2020},
author = {Dance, A},
title = {Studying life at the extremes.},
journal = {Nature},
volume = {587},
number = {7832},
pages = {165-166},
doi = {10.1038/d41586-020-03055-0},
pmid = {33139946},
issn = {1476-4687},
mesh = {Bacteria/*cytology/*genetics/isolation &amp; purification ; CRISPR-Cas Systems ; Cell Division ; Clostridium thermocellum/genetics ; DNA Methylation ; *Extreme Environments ; Gene Editing/methods ; Genes, Bacterial/genetics ; Halobacterium salinarum/cytology ; Haloferax volcanii/cytology/genetics/metabolism ; *Laboratories ; Microbiological Techniques/*methods ; Microscopy/instrumentation/methods ; *Research Design ; *Research Personnel ; Sulfolobus acidocaldarius/cytology ; },
}
@article {pmid33139880,
year = {2021},
author = {Martínez Arbas, S and Narayanasamy, S and Herold, M and Lebrun, LA and Hoopmann, MR and Li, S and Lam, TJ and Kunath, BJ and Hicks, ND and Liu, CM and Price, LB and Laczny, CC and Gillece, JD and Schupp, JM and Keim, PS and Moritz, RL and Faust, K and Tang, H and Ye, Y and Skupin, A and May, P and Muller, EEL and Wilmes, P},
title = {Roles of bacteriophages, plasmids and CRISPR immunity in microbial community dynamics revealed using time-series integrated meta-omics.},
journal = {Nature microbiology},
volume = {6},
number = {1},
pages = {123-135},
pmid = {33139880},
issn = {2058-5276},
support = {PHD-2014-1/7934898//Fonds National de la Recherche Luxembourg (National Research Fund)/ ; C15/SR/10404839//Fonds National de la Recherche Luxembourg (National Research Fund)/ ; ATTRACT (A09/03)//Fonds National de la Recherche Luxembourg (National Research Fund)/ ; CORE/15/BM/10404093//Fonds National de la Recherche Luxembourg (National Research Fund)/ ; CORE/17/SM/11689322//Fonds National de la Recherche Luxembourg (National Research Fund)/ ; PRIDE15/10907093//Fonds National de la Recherche Luxembourg (National Research Fund)/ ; },
abstract = {Viruses and plasmids (invasive mobile genetic elements (iMGEs)) have important roles in shaping microbial communities, but their dynamic interactions with CRISPR-based immunity remain unresolved. We analysed generation-resolved iMGE-host dynamics spanning one and a half years in a microbial consortium from a biological wastewater treatment plant using integrated meta-omics. We identified 31 bacterial metagenome-assembled genomes encoding complete CRISPR-Cas systems and their corresponding iMGEs. CRISPR-targeted plasmids outnumbered their bacteriophage counterparts by at least fivefold, highlighting the importance of CRISPR-mediated defence against plasmids. Linear modelling of our time-series data revealed that the variation in plasmid abundance over time explained more of the observed community dynamics than phages. Community-scale CRISPR-based plasmid-host and phage-host interaction networks revealed an increase in CRISPR-mediated interactions coinciding with a decrease in the dominant 'Candidatus Microthrix parvicella' population. Protospacers were enriched in sequences targeting genes involved in the transmission of iMGEs. Understanding the factors shaping the fitness of specific populations is necessary to devise control strategies for undesirable species and to predict or explain community-wide phenotypes.},
}
@article {pmid33139742,
year = {2020},
author = {Gasiunas, G and Young, JK and Karvelis, T and Kazlauskas, D and Urbaitis, T and Jasnauskaite, M and Grusyte, MM and Paulraj, S and Wang, PH and Hou, Z and Dooley, SK and Cigan, M and Alarcon, C and Chilcoat, ND and Bigelyte, G and Curcuru, JL and Mabuchi, M and Sun, Z and Fuchs, RT and Schildkraut, E and Weigele, PR and Jack, WE and Robb, GB and Venclovas, Č and Siksnys, V},
title = {A catalogue of biochemically diverse CRISPR-Cas9 orthologs.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5512},
pmid = {33139742},
issn = {2041-1723},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Computational Biology ; DNA Cleavage ; Gene Editing/*methods ; RNA, Guide/*genetics/metabolism ; Sequence Homology, Nucleic Acid ; },
abstract = {Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.},
}
@article {pmid33139725,
year = {2020},
author = {Dougherty, GW and Mizuno, K and Nöthe-Menchen, T and Ikawa, Y and Boldt, K and Ta-Shma, A and Aprea, I and Minegishi, K and Pang, YP and Pennekamp, P and Loges, NT and Raidt, J and Hjeij, R and Wallmeier, J and Mussaffi, H and Perles, Z and Elpeleg, O and Rabert, F and Shiratori, H and Letteboer, SJ and Horn, N and Young, S and Strünker, T and Stumme, F and Werner, C and Olbrich, H and Takaoka, K and Ide, T and Twan, WK and Biebach, L and Große-Onnebrink, J and Klinkenbusch, JA and Praveen, K and Bracht, DC and Höben, IM and Junger, K and Gützlaff, J and Cindrić, S and Aviram, M and Kaiser, T and Memari, Y and Dzeja, PP and Dworniczak, B and Ueffing, M and Roepman, R and Bartscherer, K and Katsanis, N and Davis, EE and Amirav, I and Hamada, H and Omran, H},
title = {CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5520},
pmid = {33139725},
issn = {2041-1723},
support = {R01 DK072301/DK/NIDDK NIH HHS/United States ; },
mesh = {Adenine Nucleotides/*metabolism ; Adolescent ; Adult ; Animals ; Asthenozoospermia/*genetics/pathology ; Axoneme/ultrastructure ; CRISPR-Cas Systems/genetics ; Cilia/metabolism/ultrastructure ; Cytoskeletal Proteins/*deficiency/genetics ; DNA Mutational Analysis ; Disease Models, Animal ; Epididymis/pathology ; Female ; Flagella/metabolism/ultrastructure ; Humans ; Loss of Function Mutation ; Male ; Mice ; Mice, Knockout ; Middle Aged ; Planarians/cytology/genetics/metabolism ; Respiratory Mucosa/cytology/pathology ; Situs Inversus/diagnostic imaging/*genetics/pathology ; Sperm Motility/genetics ; Tomography, X-Ray Computed ; Whole Exome Sequencing ; },
abstract = {Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.},
}
@article {pmid33137817,
year = {2020},
author = {Yan, J and Xue, D and Chuai, G and Gao, Y and Zhang, G and Liu, Q},
title = {Benchmarking and integrating genome-wide CRISPR off-target detection and prediction.},
journal = {Nucleic acids research},
volume = {48},
number = {20},
pages = {11370-11379},
pmid = {33137817},
issn = {1362-4962},
mesh = {Algorithms ; Benchmarking ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Computer Simulation ; Databases, Genetic ; Gene Editing/*methods ; Gene Knockout Techniques ; Genome ; Genomics/*methods ; Humans ; Models, Molecular ; RNA, Guide ; Whole Genome Sequencing ; },
abstract = {Systematic evaluation of genome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) off-target profiles is a fundamental step for the successful application of the CRISPR system to clinical therapies. Many experimental techniques and in silico tools have been proposed for detecting and predicting genome-wide CRISPR off-target profiles. These techniques and tools, however, have not been systematically benchmarked. A comprehensive benchmark study and an integrated strategy that takes advantage of the currently available tools to improve predictions of genome-wide CRISPR off-target profiles are needed. We focused on the specificity of the traditional CRISPR SpCas9 system for gene knockout. First, we benchmarked 10 available genome-wide off-target cleavage site (OTS) detection techniques with the published OTS detection datasets. Second, taking the datasets generated from OTS detection techniques as the benchmark datasets, we benchmarked 17 available in silico genome-wide OTS prediction tools to evaluate their genome-wide CRISPR off-target prediction performances. Finally, we present the first one-stop integrated Genome-Wide Off-target cleavage Search platform (iGWOS) that was specifically designed for the optimal genome-wide OTS prediction by integrating the available OTS prediction algorithms with an AdaBoost ensemble framework.},
}
@article {pmid33137193,
year = {2020},
author = {Wang, J and Dai, W and Li, J and Li, Q and Xie, R and Zhang, Y and Stubenrauch, C and Lithgow, T},
title = {AcrHub: an integrative hub for investigating, predicting and mapping anti-CRISPR proteins.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa951},
pmid = {33137193},
issn = {1362-4962},
abstract = {Anti-CRISPR (Acr) proteins naturally inhibit CRISPR-Cas adaptive immune systems across bacterial and archaeal domains of life. This emerging field has caused a paradigm shift in the way we think about the CRISPR-Cas system, and promises a number of useful applications from gene editing to phage therapy. As the number of verified and predicted Acrs rapidly expands, few online resources have been developed to deal with this wealth of information. To overcome this shortcoming, we developed AcrHub, an integrative database to provide an all-in-one solution for investigating, predicting and mapping Acr proteins. AcrHub catalogs 339 non-redundant experimentally validated Acrs and over 70 000 predicted Acrs extracted from genome sequence data from a diverse range of prokaryotic organisms and their viruses. It integrates state-of-the-art predictors to predict potential Acrs, and incorporates three analytical modules: similarity analysis, phylogenetic analysis and homology network analysis, to analyze their relationships with known Acrs. By interconnecting all modules as a platform, AcrHub presents enriched and in-depth analysis of known and potential Acrs and therefore provides new and exciting insights into the future of Acr discovery and validation. AcrHub is freely available at http://pacrispr.erc.monash.edu/AcrHub/.},
}
@article {pmid33135430,
year = {2020},
author = {Straiton, J},
title = {CRISPR vs COVID-19: how can gene editing help beat a virus?.},
journal = {BioTechniques},
volume = {69},
number = {5},
pages = {327-329},
doi = {10.2144/btn-2020-0145},
pmid = {33135430},
issn = {1940-9818},
mesh = {Antiviral Agents/therapeutic use ; Betacoronavirus/genetics ; COVID-19 ; *CRISPR-Cas Systems ; *Coronavirus Infections/diagnosis/drug therapy/therapy ; *Gene Editing ; Gene Targeting ; Humans ; Molecular Diagnostic Techniques/methods ; *Molecular Targeted Therapy ; *Pandemics ; *Pneumonia, Viral/diagnosis/therapy ; SARS-CoV-2 ; },
abstract = {[Formula: see text] Known to be a sturdy weapon in a scientist's arsenal, how has the gene editing tool CRISPR been applied in the fight against COVID-19?},
}
@article {pmid33134311,
year = {2020},
author = {Konwarh, R},
title = {Can CRISPR/Cas Technology Be a Felicitous Stratagem Against the COVID-19 Fiasco? Prospects and Hitches.},
journal = {Frontiers in molecular biosciences},
volume = {7},
number = {},
pages = {557377},
pmid = {33134311},
issn = {2296-889X},
abstract = {The current global debacle of COVID-19, spelled by SARS-CoV-2 needs no elaboration. With incessant and constantly clambering number of deaths across various nations, the need of the hour is to develop readily deployable, fast, affordable detection assays and kits, yielding precise and consistent results as well as timely availability of efficacious anti-SARS-CoV-2 strategies to contain it. Conventionally employed real time PCR based technique for detection of the virus suffers from a couple of handicaps. Amongst other approaches, CRISPR based technology has ushered in new hopes. Recent efforts have been directed toward developing CRISPR/Cas based low-cost, rapid detection methods as well as development of one-pot assay platforms. The plausible application of CRISPR-Cas system to counteract the viral assault has also been assessed. The write up in this article mirrors the current status, the prospects and the practical snags of CRISPR/Cas technology for the detection and inactivation of the novel corona virus, SARS-CoV-2.},
}
@article {pmid33134100,
year = {2020},
author = {Lomov, NA and Viushkov, VS and Zamalutdinov, AV and Sboeva, MD and Rubtsov, MA},
title = {Direct ENIT: An easy and reliable tool for gRNA efficacy verification by tracking induced chromosomal translocation.},
journal = {MethodsX},
volume = {7},
number = {},
pages = {101104},
pmid = {33134100},
issn = {2215-0161},
abstract = {CRISPR/Cas systems (Clustered regularly interspaced palindromic repeats / CRISPR-associated) are rapidly becoming a commonplace and popular tool for gene editing in research and clinical contexts. However, the quality of CRISPR/Cas experiments depends heavily on the guide RNA (gRNA) design; therefore, a reliable, easy, and rapid method for verifying gRNA cleavage efficacy is necessary. Engineered nuclease-induced translocations (ENIT) are an easy and cost-efficient method for the verification of gRNA efficacy, which involves tracking induced chromosomal mutations, using polymerase chain reaction (PCR). We have customized this method using both direct PCR and nested PCR approaches and have been able to reduce the sample preparation time. We present a simple and reliable gRNA testing approach that requires no specific enzymes or equipment.•The approach requires only routinely used enzymes and equipment.•Cost- and time-efficient, requiring approximately 30 min for PCR sample preparation, without requiring DNA purification.•High sensitivity, with induced translocation detected in 100 of 10,000 cells in the general population.},
}
@article {pmid33132845,
year = {2020},
author = {Li, F and Wing, K and Wang, JH and Luu, CD and Bender, JA and Chen, J and Wang, Q and Lu, Q and Nguyen Tran, MT and Young, KM and Wong, RCB and Pébay, A and Cook, AL and Hung, SSC and Liu, GS and Hewitt, AW},
title = {Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing.},
journal = {Frontiers in cellular neuroscience},
volume = {14},
number = {},
pages = {570917},
pmid = {33132845},
issn = {1662-5102},
abstract = {CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vivo.},
}
@article {pmid33132758,
year = {2020},
author = {Miri, SM and Tafsiri, E and Cho, WCS and Ghaemi, A},
title = {Correction to: CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy.},
journal = {Cancer cell international},
volume = {20},
number = {},
pages = {521},
pmid = {33132758},
issn = {1475-2867},
abstract = {[This corrects the article DOI: 10.1186/s12935-020-01546-8.].},
}
@article {pmid33128871,
year = {2020},
author = {Takahashi, N and Cho, P and Selfors, LM and Kuiken, HJ and Kaul, R and Fujiwara, T and Harris, IS and Zhang, T and Gygi, SP and Brugge, JS},
title = {3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2 as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis.},
journal = {Molecular cell},
volume = {80},
number = {5},
pages = {828-844.e6},
doi = {10.1016/j.molcel.2020.10.010},
pmid = {33128871},
issn = {1097-4164},
support = {R01 GM067945/GM/NIGMS NIH HHS/United States ; },
mesh = {A549 Cells ; *CRISPR-Cas Systems ; *Cell Culture Techniques ; *Cell Proliferation ; *Ferroptosis ; Humans ; NF-E2-Related Factor 2/genetics/*metabolism ; Neoplasm Proteins/genetics/*metabolism ; Neoplasms/genetics/*metabolism/pathology ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics/metabolism ; Spheroids, Cellular/*metabolism/pathology ; TOR Serine-Threonine Kinases/genetics/metabolism ; },
abstract = {Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.},
}
@article {pmid33127914,
year = {2020},
author = {Liu, Y and Huang, W and Cai, Z},
title = {Synthesizing AND gate minigene circuits based on CRISPReader for identification of bladder cancer cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5486},
pmid = {33127914},
issn = {2041-1723},
mesh = {Animals ; Apoptosis ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Epithelial Cells ; Female ; Gene Editing/methods ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks/*genetics ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Synthetic Biology ; Urinary Bladder ; Urinary Bladder Neoplasms/*diagnosis/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {The logical AND gate gene circuit based on the CRISPR-Cas9 system can distinguish bladder cancer cells from normal bladder epithelial cells. However, the layered artificial gene circuits have the problems of high complexity, difficulty in accurately predicting the behavior, and excessive redundancy, which cannot be applied to clinical translation. Here, we construct minigene circuits based on the CRISPReader, a technology used to control promoter-less gene expression in a robust manner. The minigene circuits significantly induce robust gene expression output in bladder cancer cells, but have nearly undetectable gene expression in normal bladder epithelial cells. The minigene circuits show a higher capability for cancer identification and intervention when compared with traditional gene circuits, and could be used for in vivo cancer gene therapy using the all-in-one AAV vector. This approach expands the design ideas and concepts of gene circuits in medical synthetic biology.},
}
@article {pmid33126728,
year = {2020},
author = {Ophinni, Y and Miki, S and Hayashi, Y and Kameoka, M},
title = {Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape.},
journal = {Viruses},
volume = {12},
number = {11},
pages = {},
pmid = {33126728},
issn = {1999-4915},
abstract = {HIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA). Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape. The T cell line were transduced with single and multiple gRNAs targeting HIV-1 tat and rev using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1NL4-3 challenge in vitro. Viral p24 rebound was observed for almost all gRNAs, but multiplexing three tat-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape. Multiplexed tat gRNAs inhibited acute viral replication in the 2nd round of infection, abolished cell-associated transmission to unprotected T cells, and maintained protection through 45 days, post-infection (dpi) after a higher dose of HIV-1 infection. Finally, we describe here for the first time the assembly of all-in-one lentiviral vectors containing three and six gRNAs targeting tat and rev. A single-vector tat-targeting construct shows non-inferiority to the tat-targeting multi-vector in low-dose HIV-1 infection. We conclude that Cas9-induced, DNA repair-mediated mutations in tat are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of tat further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure.},
}
@article {pmid33126177,
year = {2021},
author = {Wang, R and Qian, C and Pang, Y and Li, M and Yang, Y and Ma, H and Zhao, M and Qian, F and Yu, H and Liu, Z and Ni, T and Zheng, Y and Wang, Y},
title = {opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {172},
number = {},
pages = {112766},
doi = {10.1016/j.bios.2020.112766},
pmid = {33126177},
issn = {1873-4235},
mesh = {Base Sequence ; COVID-19/*diagnosis/virology ; COVID-19 Nucleic Acid Testing/instrumentation/*methods/statistics &amp; numerical data ; *CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/instrumentation/*methods/statistics &amp; numerical data ; Nucleic Acid Amplification Techniques/instrumentation/*methods/statistics &amp; numerical data ; Pandemics ; RNA, Viral/genetics ; SARS-CoV-2/*genetics/isolation &amp; purification ; Sensitivity and Specificity ; },
abstract = {The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named &quot;opvCRISPR&quot; by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.},
}
@article {pmid33124980,
year = {2020},
author = {Stringer, AM and Baniulyte, G and Lasek-Nesselquist, E and Seed, KD and Wade, JT},
title = {Transcription termination and antitermination of bacterial CRISPR arrays.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
pmid = {33124980},
issn = {2050-084X},
support = {R01 GM122836/GM/NIGMS NIH HHS/United States ; R21 AI126416/AI/NIAID NIH HHS/United States ; R01 AI127652/AI/NIAID NIH HHS/United States ; },
abstract = {A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences ('repeats') interspersed with short, variable sequences ('spacers'). CRISPR arrays are transcribed and processed into individual CRISPR RNAs that each include a single spacer, and direct Cas proteins to complementary sequences in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that Rho can prematurely terminate transcription of bacterial CRISPR arrays, and we identify a widespread antitermination mechanism that antagonizes Rho to facilitate complete transcription of CRISPR arrays. Thus, our data highlight the importance of transcription termination and antitermination in the evolution of bacterial CRISPR-Cas systems.},
}
@article {pmid33123803,
year = {2020},
author = {Khalil, AM},
title = {The genome editing revolution: review.},
journal = {Journal, genetic engineering &amp; biotechnology},
volume = {18},
number = {1},
pages = {68},
pmid = {33123803},
issn = {2090-5920},
abstract = {BACKGROUND: Development of efficient strategies has always been one of the great perspectives for biotechnologists. During the last decade, genome editing of different organisms has been a fast advancing field and therefore has received a lot of attention from various researchers comprehensively reviewing latest achievements and offering opinions on future directions. This review presents a brief history, basic principles, advantages and disadvantages, as well as various aspects of each genome editing technology including the modes, applications, and challenges that face delivery of gene editing components.<br><br>MAIN BODY: Genetic modification techniques cover a wide range of studies, including the generation of transgenic animals, functional analysis of genes, model development for diseases, or drug development. The delivery of certain proteins such as monoclonal antibodies, enzymes, and growth hormones has been suffering from several obstacles because of their large size. These difficulties encouraged scientists to explore alternative approaches, leading to the progress in gene editing. The distinguished efforts and enormous experimentation have now been able to introduce methodologies that can change the genetic constitution of the living cell. The genome editing strategies have evolved during the last three decades, and nowadays, four types of &quot;programmable&quot; nucleases are available in this field: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system. Each group has its own characteristics necessary for researchers to select the most suitable method for gene editing tool for a range of applications. Genome engineering/editing technology will revolutionize the creation of precisely manipulated genomes of cells or organisms in order to modify a specific characteristic. Of the potential applications are those in human health and agriculture. Introducing constructs into target cells or organisms is the key step in genome engineering.<br><br>CONCLUSIONS: Despite the success already achieved, the genome editing techniques are still suffering certain difficulties. Challenges must be overcome before the full potential of genome editing can be realized.},
}
@article {pmid33123724,
year = {2020},
author = {Zhao, J and Inomata, R and Kato, Y and Miyagishi, M},
title = {Development of aptamer-based inhibitors for CRISPR/Cas system.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa865},
pmid = {33123724},
issn = {1362-4962},
abstract = {The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.},
}
@article {pmid33123486,
year = {2020},
author = {Zhang, Y and Zhao, G and Ahmed, FYH and Yi, T and Hu, S and Cai, T and Liao, Q},
title = {In silico Method in CRISPR/Cas System: An Expedite and Powerful Booster.},
journal = {Frontiers in oncology},
volume = {10},
number = {},
pages = {584404},
pmid = {33123486},
issn = {2234-943X},
abstract = {The CRISPR/Cas system has stood in the center of attention in the last few years as a revolutionary gene editing tool with a wide application to investigate gene functions. However, the labor-intensive workflow requires a sophisticated pre-experimental and post-experimental analysis, thus becoming one of the hindrances for the further popularization of practical applications. Recently, the increasing emergence and advancement of the in silico methods play a formidable role to support and boost experimental work. However, various tools based on distinctive design principles and frameworks harbor unique characteristics that are likely to confuse users about how to choose the most appropriate one for their purpose. In this review, we will present a comprehensive overview and comparisons on the in silico methods from the aspects of CRISPR/Cas system identification, guide RNA design, and post-experimental assistance. Furthermore, we establish the hypotheses in light of the new trends around the technical optimization and hope to provide significant clues for future tools development.},
}
@article {pmid33122438,
year = {2020},
author = {Westra, ER and Levin, BR},
title = {It is unclear how important CRISPR-Cas systems are for protecting natural populations of bacteria against infections by mobile genetic elements.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {45},
pages = {27777-27785},
pmid = {33122438},
issn = {1091-6490},
support = {R01 GM091875/GM/NIGMS NIH HHS/United States ; R35 GM136407/GM/NIGMS NIH HHS/United States ; BB/N017412/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Articles on CRISPR commonly open with some variant of the phrase &quot;these short palindromic repeats and their associated endonucleases (Cas) are an adaptive immune system that exists to protect bacteria and archaea from viruses and infections with other mobile genetic elements.&quot; There is an abundance of genomic data consistent with the hypothesis that CRISPR plays this role in natural populations of bacteria and archaea, and experimental demonstrations with a few species of bacteria and their phage and plasmids show that CRISPR-Cas systems can play this role in vitro. Not at all clear are the ubiquity, magnitude, and nature of the contribution of CRISPR-Cas systems to the ecology and evolution of natural populations of microbes and the strength of selection mediated by different types of phage and plasmids to the evolution and maintenance of CRISPR-Cas systems. In this perspective, with the aid of heuristic mathematical-computer simulation models, we explore the a priori conditions under which exposure to lytic and temperate phage and conjugative plasmids will select for and maintain CRISPR-Cas systems in populations of bacteria and archaea. We review the existing literature addressing these ecological and evolutionary questions and highlight the experimental and other evidence needed to fully understand the conditions responsible for the evolution and maintenance of CRISPR-Cas systems and the contribution of these systems to the ecology and evolution of bacteria, archaea, and the mobile genetic elements that infect them.},
}
@article {pmid33121109,
year = {2020},
author = {Dolskiy, AA and Grishchenko, IV and Yudkin, DV},
title = {Cell Cultures for Virology: Usability, Advantages, and Prospects.},
journal = {International journal of molecular sciences},
volume = {21},
number = {21},
pages = {},
pmid = {33121109},
issn = {1422-0067},
support = {075-15-2019-1665//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.},
}
@article {pmid33121045,
year = {2020},
author = {Zamai, L},
title = {Unveiling Human Non-Random Genome Editing Mechanisms Activated in Response to Chronic Environmental Changes: I. Where Might These Mechanisms Come from and What Might They Have Led To?.},
journal = {Cells},
volume = {9},
number = {11},
pages = {},
pmid = {33121045},
issn = {2073-4409},
abstract = {This article challenges the notion of the randomness of mutations in eukaryotic cells by unveiling stress-induced human non-random genome editing mechanisms. To account for the existence of such mechanisms, I have developed molecular concepts of the cell environment and cell environmental stressors and, making use of a large quantity of published data, hypothesised the origin of some crucial biological leaps along the evolutionary path of life on Earth under the pressure of natural selection, in particular, (1) virus-cell mating as a primordial form of sexual recombination and symbiosis; (2) Lamarckian CRISPR-Cas systems; (3) eukaryotic gene development; (4) antiviral activity of retrotransposon-guided mutagenic enzymes; and finally, (5) the exaptation of antiviral mutagenic mechanisms to stress-induced genome editing mechanisms directed at &quot;hyper-transcribed&quot; endogenous genes. Genes transcribed at their maximum rate (hyper-transcribed), yet still unable to meet new chronic environmental demands generated by &quot;pollution&quot;, are inadequate and generate more and more intronic retrotransposon transcripts. In this scenario, RNA-guided mutagenic enzymes (e.g., Apolipoprotein B mRNA editing catalytic polypeptide-like enzymes, APOBECs), which have been shown to bind to retrotransposon RNA-repetitive sequences, would be surgically targeted by intronic retrotransposons on opened chromatin regions of the same &quot;hyper-transcribed&quot; genes. RNA-guided mutagenic enzymes may therefore &quot;Lamarkianly&quot; generate single nucleotide polymorphisms (SNP) and gene copy number variations (CNV), as well as transposon transposition and chromosomal translocations in the restricted areas of hyper-functional and inadequate genes, leaving intact the rest of the genome. CNV and SNP of hyper-transcribed genes may allow cells to surgically explore a new fitness scenario, which increases their adaptability to stressful environmental conditions. Like the mechanisms of immunoglobulin somatic hypermutation, non-random genome editing mechanisms may generate several cell mutants, and those codifying for the most environmentally adequate proteins would have a survival advantage and would therefore be Darwinianly selected. Non-random genome editing mechanisms represent tools of evolvability leading to organismal adaptation including transgenerational non-Mendelian gene transmission or to death of environmentally inadequate genomes. They are a link between environmental changes and biological novelty and plasticity, finally providing a molecular basis to reconcile gene-centred and &quot;ecological&quot; views of evolution.},
}
@article {pmid33117361,
year = {2020},
author = {Pavlovic, K and Tristán-Manzano, M and Maldonado-Pérez, N and Cortijo-Gutierrez, M and Sánchez-Hernández, S and Justicia-Lirio, P and Carmona, MD and Herrera, C and Martin, F and Benabdellah, K},
title = {Using Gene Editing Approaches to Fine-Tune the Immune System.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {570672},
pmid = {33117361},
issn = {1664-3224},
abstract = {Genome editing technologies not only provide unprecedented opportunities to study basic cellular system functionality but also improve the outcomes of several clinical applications. In this review, we analyze various gene editing techniques used to fine-tune immune systems from a basic research and clinical perspective. We discuss recent advances in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We also discuss the use of programmable nucleases and their derivative reagents such as base editing tools to engineer immune cells via gene disruption, insertion, and rewriting of T cells and other immune components, such natural killers (NKs) and hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric antigen receptors (CARs), we describe how different gene editing tools enable healthy donor cells to be used in CAR T therapy instead of autologous cells without risking graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs and instant treatment availability for patients. We pay particular attention to the delivery of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral damage and increases therapeutic effectiveness. Finally, we review creative innovations, including immune system repurposing, that facilitate safe and efficient genome surgery within the framework of clinical cancer immunotherapies.},
}
@article {pmid33117331,
year = {2020},
author = {Azangou-Khyavy, M and Ghasemi, M and Khanali, J and Boroomand-Saboor, M and Jamalkhah, M and Soleimani, M and Kiani, J},
title = {CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer.},
journal = {Frontiers in immunology},
volume = {11},
number = {},
pages = {2062},
pmid = {33117331},
issn = {1664-3224},
abstract = {The clustered regularly interspaced short palindromic repeats system has demonstrated considerable advantages over other nuclease-based genome editing tools due to its high accuracy, efficiency, and strong specificity. Given that cancer is caused by an excessive accumulation of mutations that lead to the activation of oncogenes and inactivation of tumor suppressor genes, the CRISPR/Cas9 system is a therapy of choice for tumor genome editing and treatment. In defining its superior use, we have reviewed the novel applications of the CRISPR genome editing tool in discovering, sorting, and prioritizing targets for subsequent interventions, and passing different hurdles of cancer treatment such as epigenetic alterations and drug resistance. Moreover, we have reviewed the breakthroughs precipitated by the CRISPR system in the field of cancer immunotherapy, such as identification of immune system-tumor interplay, production of universal Chimeric Antigen Receptor T cells, inhibition of immune checkpoint inhibitors, and Oncolytic Virotherapy. The existing challenges and limitations, as well as the prospects of CRISPR based systems, are also discussed.},
}
@article {pmid33114339,
year = {2020},
author = {Bröker, JN and Müller, B and Prüfer, D and Schulze Gronover, C},
title = {Combinatorial Metabolic Engineering in Saccharomyces cerevisiae for the Enhanced Production of the FPP-Derived Sesquiterpene Germacrene.},
journal = {Bioengineering (Basel, Switzerland)},
volume = {7},
number = {4},
pages = {},
pmid = {33114339},
issn = {2306-5354},
support = {031B0014D//Bundesministerium für Wissenschaft und Forschung/ ; },
abstract = {Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.},
}
@article {pmid33113496,
year = {2020},
author = {Malone, LM and Birkholz, N and Fineran, PC},
title = {Conquering CRISPR: how phages overcome bacterial adaptive immunity.},
journal = {Current opinion in biotechnology},
volume = {68},
number = {},
pages = {30-36},
doi = {10.1016/j.copbio.2020.09.008},
pmid = {33113496},
issn = {1879-0429},
abstract = {The rise of antibiotic-resistant bacteria has led to renewed interest in the use of their natural enemies, phages, for the prevention and treatment of infections. However, phage therapy requires detailed knowledge of the interactions between these entities. Bacteria defend themselves against phage predation with a large repertoire of defences. Among these, CRISPR-Cas systems stand out due to their adaptive character, mechanistic complexity and diversity, and present a significant hurdle for phage infection. Here, we provide an overview of how phages can circumvent CRISPR-Cas defence, ranging from target sequence mutations and DNA modifications to anti-CRISPR proteins and nucleus-like protective structures. An in-depth understanding of these phage evasion strategies is crucial for the successful development of phage therapy applications.},
}
@article {pmid33111087,
year = {2020},
author = {Xu, Z and Li, Y and Yan, A},
title = {Repurposing the Native Type I-F CRISPR-Cas System in Pseudomonas aeruginosa for Genome Editing.},
journal = {STAR protocols},
volume = {1},
number = {1},
pages = {100039},
pmid = {33111087},
issn = {2666-1667},
abstract = {Repurposing the broadly distributed native CRISPR-Cas systems in prokaryotes for genome editing is emerging as a new strategy for genetic manipulations. We recently reported the establishment of a single plasmid-mediated, one-step genome-editing technique in a multidrug-resistant genotype of the opportunistic pathogen Pseudomonas aeruginosa by harnessing its endogenous type I-F CRISPR-Cas system. The platform is readily applicable in additional type I-F CRISPR-containing clinical and environmental P. aeruginosa isolates. Herein, we provide the detailed protocol for the methodology. For complete details on the establishment and exploitation of this protocol, please refer to Xu et al. (2019).},
}
@article {pmid33109771,
year = {2020},
author = {Sun, H and Li, Q and Yin, G and Ding, X and Xie, J},
title = {Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes.},
journal = {Aging},
volume = {12},
number = {20},
pages = {20432-20444},
pmid = {33109771},
issn = {1945-4589},
abstract = {In human macrophages and monocytes, lipopolysaccharide (LPS) induces nuclear factor kappa B (NFκB) activation and pro-inflammatory cytokines production. We tested the possible involvement of Ku70 and Ku80 in the process. In THP-1 macrophages and primary human peripheral blood mononuclear cells (PBMCs), shRNA-induced double knockdown of Ku70 and Ku80 potently inhibited LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Additionally, we developed CRISPR/Cas-9 gene-editing methods to knockout both Ku70 and Ku80 in THP-1 cells and PBMCs. Double knockout (DKO) largely inhibited LPS-induced pro-inflammatory cytokines production. Conversely, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 enhanced the pro-inflammatory cytokines production by LPS. Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in the nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced NFκB activation was inhibited by Ku70 plus Ku80 double knockdown or DKO. It was however enhanced with Ku70 and Ku80 overexpression. Together, Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response in THP-1 cells and human PBMCs.},
}
@article {pmid33104071,
year = {2020},
author = {Xu, D and Mondol, PC and Uzair, M and Tucker, MR and Zhang, D},
title = {Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {164},
pages = {},
doi = {10.3791/61665},
pmid = {33104071},
issn = {1940-087X},
mesh = {Agrobacterium/*genetics ; Base Sequence ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genotype ; Mutagenesis/genetics ; Mutation/genetics ; Oryza/*genetics/*physiology ; Phenotype ; Plants, Genetically Modified/genetics ; Pollen/*genetics/*growth &amp; development ; Reproduction/physiology ; Seedlings/genetics ; Tissue Culture Techniques ; *Transformation, Genetic ; },
abstract = {Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.},
}
@article {pmid33102460,
year = {2020},
author = {Yu, L and Marchisio, MA},
title = {Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {575393},
pmid = {33102460},
issn = {2296-4185},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins), a prokaryotic RNA-mediated adaptive immune system, has been repurposed for gene editing and synthetic gene circuit construction both in bacterial and eukaryotic cells. In the last years, the emergence of the anti-CRISPR proteins (Acrs), which are natural OFF-switches for CRISPR-Cas, has provided a new means to control CRISPR-Cas activity and promoted a further development of CRISPR-Cas-based biotechnological toolkits. In this review, we focus on type I and type V-A anti-CRISPR proteins. We first narrate Acrs discovery and analyze their inhibitory mechanisms from a structural perspective. Then, we describe their applications in gene editing and transcription regulation. Finally, we discuss the potential future usage-and corresponding possible challenges-of these two kinds of anti-CRISPR proteins in eukaryotic synthetic gene circuits.},
}
@article {pmid33102417,
year = {2020},
author = {Almeida, LM and Gaca, A and Bispo, PM and Lebreton, F and Saavedra, JT and Silva, RA and Basílio-Júnior, ID and Zorzi, FM and Filsner, PH and Moreno, AM and Gilmore, MS},
title = {Coexistence of the Oxazolidinone Resistance-Associated Genes cfr and optrA in Enterococcus faecalis From a Healthy Piglet in Brazil.},
journal = {Frontiers in public health},
volume = {8},
number = {},
pages = {518},
pmid = {33102417},
issn = {2296-2565},
support = {P01 AI083214/AI/NIAID NIH HHS/United States ; },
abstract = {Oxazolidinones are one of the most important antimicrobials potentially active against glycopeptide- and β-lactam-resistant Gram-positive pathogens. Linezolid-the first oxazolidinone to be approved for clinical use in 2000 by the US Food and Drug Administration-and the newer molecule in the class, tedizolid, inhibit protein synthesis by suppressing the formation of the 70S ribosomal complex in bacteria. Over the past two decades, transferable oxazolidinone resistance genes, in particular cfr and optrA, have been identified in Firmicutes isolated from healthcare-related infections, livestock, and the environment. Our goals in this study were to investigate the genetic contexts and the transferability of the cfr and optrA genes and examine genomic features, such as antimicrobial resistance genes, plasmid incompatibility types, and CRISPR-Cas defenses of a linezolid-resistant Enterococcus faecalis isolated in feces from a healthy pig during an antimicrobial surveillance program for animal production in Brazil. The cfr gene was found to be integrated into a transposon-like structure of 7,759 nt flanked by IS1216E and capable of excising and circularizing, distinguishing it from known genetic contexts for cfr in Enterococcus spp., while optrA was inserted into an Inc18 broad host-range plasmid of >58 kb. Conjugal transfer of cfr and optrA was shown by filter mating. The coexistence of cfr and optrA in an E. faecalis isolated from a healthy nursery pig highlights the need for monitoring the use of antibiotics in the Brazilian swine production system for controlling spread and proliferation of antibiotic resistance.},
}
@article {pmid33097793,
year = {2020},
author = {Matsumoto, D and Tamamura, H and Nomura, W},
title = {A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {601},
pmid = {33097793},
issn = {2399-3642},
support = {JP25410171//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16K01931//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP17J08531//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24119506//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP26119703//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16H01420//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
abstract = {The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.},
}
@article {pmid33097693,
year = {2020},
author = {Schene, IF and Joore, IP and Oka, R and Mokry, M and van Vugt, AHM and van Boxtel, R and van der Doef, HPJ and van der Laan, LJW and Verstegen, MMA and van Hasselt, PM and Nieuwenhuis, EES and Fuchs, SA},
title = {Prime editing for functional repair in patient-derived disease models.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5352},
pmid = {33097693},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Copper-Transporting ATPases/genetics ; Deoxyribonuclease I/metabolism ; Diacylglycerol O-Acyltransferase/genetics ; Gene Editing/*methods ; HEK293 Cells ; Hepatolenticular Degeneration/genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; Organoids/*metabolism ; Recombinational DNA Repair ; Stem Cells ; Targeted Gene Repair/methods ; beta Catenin/*genetics ; },
abstract = {Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.},
}
@article {pmid33097498,
year = {2020},
author = {García-Moyano, A and Larsen, Ø and Gaykawad, S and Christakou, E and Boccadoro, C and Puntervoll, P and Bjerga, GEK},
title = {Fragment Exchange Plasmid Tools for CRISPR/Cas9-Mediated Gene Integration and Protease Production in Bacillus subtilis.},
journal = {Applied and environmental microbiology},
volume = {87},
number = {1},
pages = {},
pmid = {33097498},
issn = {1098-5336},
abstract = {Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool for targeted genome editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial species. A collection of vectors compatible with high-throughput (HTP) fragment exchange (FX) cloning for heterologous expression in Escherichia coli and Bacillus was previously developed. This vector catalogue was through this work supplemented with editing plasmids for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to the stable integration of the gene into the amyE locus of a seven-protease-deficient KO7 strain. The newly engineered B. subtilis strain allowed the successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering, and contribute to a cloning platform for a quick transition from HTP enzyme expression to production through the fermentation of industrially relevant B. subtilis and related strains.IMPORTANCE We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host.},
}
@article {pmid33096014,
year = {2020},
author = {Bertelsen, MB and Senissar, M and Nielsen, MH and Bisiak, F and Cunha, MV and Molinaro, AL and Daines, DA and Brodersen, DE},
title = {Structural Basis for Toxin Inhibition in the VapXD Toxin-Antitoxin System.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2020.10.002},
pmid = {33096014},
issn = {1878-4186},
abstract = {Bacterial type II toxin-antitoxin (TA) modules encode a toxic protein that downregulates metabolism and a specific antitoxin that binds and inhibits the toxin during normal growth. In non-typeable Haemophilus influenzae, a common cause of infections in humans, the vapXD locus was found to constitute a functional TA module and contribute to pathogenicity; however, the mode of action of VapD and the mechanism of inhibition by the VapX antitoxin remain unknown. Here, we report the structure of the intact H. influenzae VapXD complex, revealing an unusual 2:1 TA molecular stoichiometry where a Cas2-like homodimer of VapD binds a single VapX antitoxin. VapX consists of an oligonucleotide/oligosaccharide-binding domain that docks into an asymmetrical cavity on the toxin dimer. Structures of isolated VapD further reveal how a symmetrical toxin homodimer adapts to interacting with an asymmetrical antitoxin and suggest how a primordial TA system evolved to become part of CRISPR-Cas immunity systems.},
}
@article {pmid33095053,
year = {2020},
author = {Galizi, R and Duncan, JN and Rostain, W and Quinn, CM and Storch, M and Kushwaha, M and Jaramillo, A},
title = {Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {398-408},
doi = {10.1089/crispr.2020.0029},
pmid = {33095053},
issn = {2573-1602},
support = {BB/P020615/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/M017982/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {CRISPR guide RNAs (gRNAs) can be programmed with relative ease to allow the genetic editing of nearly any DNA or RNA sequence. Here, we propose novel molecular architectures to achieve RNA-dependent modulation of CRISPR activity in response to specific RNA molecules. We designed and tested, in both living Escherichia coli cells and cell-free assays for rapid prototyping, cis-repressed RNA-interacting guide RNA (igRNA) that switch to their active state only upon interaction with small RNA fragments or long RNA transcripts, including pathogen-derived mRNAs of medical relevance such as the human immunodeficiency virus infectivity factor. The proposed CRISPR-igRNAs are fully customizable and easily adaptable to the majority if not all the available CRISPR-Cas variants to modulate a variety of genetic functions in response to specific cellular conditions, providing orthogonal activation and increased specificity. We thereby foresee a large scope of application for therapeutic, diagnostic, and biotech applications in both prokaryotic and eukaryotic systems.},
}
@article {pmid33095052,
year = {2020},
author = {Wiegand, T and Semenova, E and Shiriaeva, A and Fedorov, I and Datsenko, K and Severinov, K and Wiedenheft, B},
title = {Reproducible Antigen Recognition by the Type I-F CRISPR-Cas System.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {378-387},
pmid = {33095052},
issn = {2573-1602},
support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-associated proteins 1 and 2 (Cas1-2) are necessary and sufficient for new spacer acquisition in some CRISPR-Cas systems (e.g., type I-E), but adaptation in other systems (e.g., type II-A) involves the crRNA-guided surveillance complex. Here we show that the type I-F Cas1-2/3 proteins are necessary and sufficient to produce low levels of spacer acquisition, but the presence of the type I-F crRNA-guided surveillance complex (Csy) improves the efficiency of adaptation and significantly increases the fidelity of protospacer adjacent motif selection. Sequences selected for integration are preferentially derived from specific regions of extrachromosomal DNA, and patterns of spacer selection are highly reproducible between independent biological replicates. This work helps define the role of the Csy complex in I-F adaptation and reveals that actively replicating mobile genetic elements have antigenic signatures that facilitate their integration during CRISPR adaptation.},
}
@article {pmid33095051,
year = {2020},
author = {Polkoff, KM and Chung, J and Simpson, SG and Gleason, K and Piedrahita, JA},
title = {In Vitro Validation of Transgene Expression in Gene-Edited Pigs Using CRISPR Transcriptional Activators.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {409-418},
pmid = {33095051},
issn = {2573-1602},
support = {R21 OD019738/OD/NIH HHS/United States ; R01 OD023138/OD/NIH HHS/United States ; },
abstract = {The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications in vitro before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.},
}
@article {pmid33095045,
year = {2020},
author = {Casas-Mollano, JA and Zinselmeier, MH and Erickson, SE and Smanski, MJ},
title = {CRISPR-Cas Activators for Engineering Gene Expression in Higher Eukaryotes.},
journal = {The CRISPR journal},
volume = {3},
number = {5},
pages = {350-364},
pmid = {33095045},
issn = {2573-1602},
support = {T32 GM008347/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas-based transcriptional activators allow genetic engineers to specifically induce expression of one or many target genes in trans. Here we review the many design variations of these versatile tools and compare their effectiveness in different eukaryotic systems. Lastly, we highlight several applications of programmable transcriptional activation to interrogate and engineer complex biological processes.},
}
@article {pmid33093796,
year = {2020},
author = {Sudheer, S and Bai, RG and Usmani, Z and Sharma, M},
title = {Insights on Engineered Microbes in Sustainable Agriculture: Biotechnological Developments and Future Prospects.},
journal = {Current genomics},
volume = {21},
number = {5},
pages = {321-333},
pmid = {33093796},
issn = {1389-2029},
abstract = {Background: Enhanced agricultural production is essential for increasing demand of the growing world population. At the same time, to combat the adverse effects caused by conventional agriculture practices to the environment along with the impact on human health and food security, a sustainable and healthy agricultural production needs to be practiced using beneficial microorganisms for enhanced yield. It is quite challenging because these microorganisms have rich biosynthetic repositories to produce biomolecules of interest; however, the intensive research in allied sectors and emerging genetic tools for improved microbial consortia are accepting new approaches that are helpful to farmers and agriculturists to meet the ever-increasing demand of sustainable food production. An important advancement is improved strain development via genetically engineered microbial systems (GEMS) as well as genetically modified microorganisms (GMOs) possessing known and upgraded functional characteristics to promote sustainable agriculture and food security. With the development of novel technologies such as DNA automated synthesis, sequencing and influential computational tools, molecular biology has entered the systems biology and synthetic biology era. More recently, CRISPR/Cas has been engineered to be an important tool in genetic engineering for various applications in the agri sector. The research in sustainable agriculture is progressing tremendously through GMOs/GEMS for their potential use in biofertilizers and as biopesticides.<br><br>Conclusion: In this review, we discuss the beneficial effects of engineered microorganisms through integrated sustainable agriculture production practices to improve the soil microbial health in order to increase crop productivity.},
}
@article {pmid33086895,
year = {2020},
author = {Vats, S and Bansal, R and Rana, N and Kumawat, S and Bhatt, V and Jadhav, P and Kale, V and Sathe, A and Sonah, H and Jugdaohsingh, R and Sharma, TR and Deshmukh, R},
title = {Unexplored nutritive potential of tomato to combat global malnutrition.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-32},
doi = {10.1080/10408398.2020.1832954},
pmid = {33086895},
issn = {1549-7852},
abstract = {Tomato, a widely consumed vegetable crop, offers a real potential to combat human nutritional deficiencies. Tomatoes are rich in micronutrients and other bioactive compounds (including vitamins, carotenoids, and minerals) that are known to be essential or beneficial for human health. This review highlights the current state of the art in the molecular understanding of the nutritional aspects, conventional and molecular breeding efforts, and biofortification studies undertaken to improve the nutritional content and quality of tomato. Transcriptomics and metabolomics studies, which offer a deeper understanding of the molecular regulation of the tomato's nutrients, are discussed. The potential uses of the wastes from the tomato processing industry (i.e., the peels and seed extracts) that are particularly rich in oils and proteins are also discussed. Recent advancements with CRISPR/Cas mediated gene-editing technology provide enormous opportunities to enhance the nutritional content of agricultural produces, including tomatoes. In this regard, genome editing efforts with respect to biofortification in the tomato plant are also discussed. The recent technological advancements and knowledge gaps described herein aim to help explore the unexplored nutritional potential of the tomato.},
}
@article {pmid33086040,
year = {2020},
author = {Vink, JNA and Brouns, SJJ and Hohlbein, J},
title = {Extracting Transition Rates in Particle Tracking Using Analytical Diffusion Distribution Analysis.},
journal = {Biophysical journal},
volume = {119},
number = {10},
pages = {1970-1983},
pmid = {33086040},
issn = {1542-0086},
abstract = {Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. The analysis of apparent diffusion coefficients in vivo, for example, enables researchers to determine whether biomolecules are moving alone, as part of a larger complex, or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models, especially for molecules that rapidly switch between different diffusional states. Here, we present analytical diffusion distribution analysis (anaDDA), a framework that allows for extracting transition rates from distributions of apparent diffusion coefficients calculated from short trajectories that feature less than 10 localizations per track. Under the assumption that the system is Markovian and diffusion is purely Brownian, we show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics, especially in the fast regime of 0.1-10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalyzed experimental data of DNA polymerase I diffusing in live Escherichia coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behavior of biomolecules in living cells.},
}
@article {pmid33085710,
year = {2020},
author = {Minguet, EG},
title = {Ares-GT: Design of guide RNAs targeting multiple genes for CRISPR-Cas experiments.},
journal = {PloS one},
volume = {15},
number = {10},
pages = {e0241001},
pmid = {33085710},
issn = {1932-6203},
mesh = {Arabidopsis/genetics ; Arabidopsis Proteins/*genetics ; *CRISPR-Cas Systems ; Programming Languages ; RNA, Guide/*genetics ; *Software ; },
abstract = {Guide RNA design for CRISPR genome editing of gene families is a challenging task as usually good candidate sgRNAs are tagged with low scores precisely because they match several locations in the genome, thus time-consuming manual evaluation of targets is required. To address this issues, I have developed ARES-GT, a Python local command line tool compatible with any operative system. ARES-GT allows the selection of candidate sgRNAs that match multiple input query sequences, in addition of candidate sgRNAs that specifically match each query sequence. It also contemplates the use of unmapped contigs apart from complete genomes thus allowing the use of any genome provided by user and being able to handle intraspecies allelic variability and individual polymorphisms. ARES-GT is available at GitHub (https://github.com/eugomin/ARES-GT.git).},
}
@article {pmid33084893,
year = {2020},
author = {Störtz, F and Minary, P},
title = {crisprSQL: a novel database platform for CRISPR/Cas off-target cleavage assays.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa885},
pmid = {33084893},
issn = {1362-4962},
abstract = {With ongoing development of the CRISPR/Cas programmable nuclease system, applications in the area of in vivo therapeutic gene editing are increasingly within reach. However, non-negligible off-target effects remain a major concern for clinical applications. Even though a multitude of off-target cleavage datasets have been published, a comprehensive, transparent overview tool has not yet been established. Here, we present crisprSQL (http://www.crisprsql.com), an interactive and bioinformatically enhanced collection of CRISPR/Cas9 off-target cleavage studies aimed at enriching the fields of cleavage profiling, gene editing safety analysis and transcriptomics. The current version of crisprSQL contains cleavage data from 144 guide RNAs on 25,632 guide-target pairs from human and rodent cell lines, with interaction-specific references to epigenetic markers and gene names. The first curated database of this standard, it promises to enhance safety quantification research, inform experiment design and fuel development of computational off-target prediction algorithms.},
}
@article {pmid33083786,
year = {2020},
author = {Shang, Z and Chan, SY and Song, Q and Li, P and Huang, W},
title = {The Strategies of Pathogen-Oriented Therapy on Circumventing Antimicrobial Resistance.},
journal = {Research (Washington, D.C.)},
volume = {2020},
number = {},
pages = {2016201},
pmid = {33083786},
issn = {2639-5274},
abstract = {The emerging antimicrobial resistance (AMR) poses serious threats to the global public health. Conventional antibiotics have been eclipsed in combating with drug-resistant bacteria. Moreover, the developing and deploying of novel antimicrobial drugs have trudged, as few new antibiotics are being developed over time and even fewer of them can hit the market. Alternative therapeutic strategies to resolve the AMR crisis are urgently required. Pathogen-oriented therapy (POT) springs up as a promising approach in circumventing antibiotic resistance. The tactic underling POT is applying antibacterial compounds or materials directly to infected regions to treat specific bacteria species or strains with goals of improving the drug efficacy and reducing nontargeting and the development of drug resistance. This review exemplifies recent trends in the development of POTs for circumventing AMR, including the adoption of antibiotic-antibiotic conjugates, antimicrobial peptides, therapeutic monoclonal antibodies, nanotechnologies, CRISPR-Cas systems, and microbiota modulations. Employing these alternative approaches alone or in combination shows promising advantages for addressing the growing clinical embarrassment of antibiotics in fighting drug-resistant bacteria.},
}
@article {pmid33082952,
year = {2020},
author = {Montenarh, M and Götz, C},
title = {Protein kinase CK2 and ion channels (Review).},
journal = {Biomedical reports},
volume = {13},
number = {6},
pages = {55},
pmid = {33082952},
issn = {2049-9434},
abstract = {Protein kinase CK2 appears as a tetramer or higher molecular weight oligomer composed of catalytic CK2α, CK2α' subunits and non-catalytic regulatory CK2β subunits or as individual subunits. It is implicated in a variety of different regulatory processes, such as Akt signalling, splicing and DNA repair within eukaryotic cells. The present review evaluates the influence of CK2 on ion channels in the plasma membrane. CK2 phosphorylates platform proteins such as calmodulin and ankyrin G, which bind to channel proteins for a physiological transport to and positioning into the membrane. In addition, CK2 directly phosphorylates a variety of channel proteins directly to regulate opening and closing of the channels. Thus, modulation of CK2 activities by specific inhibitors, by siRNA technology or by CRISPR/Cas technology has an influence on intracellular ion concentrations and thereby on cellular signalling. The physiological regulation of the intracellular ion concentration is important for cell survival and correct intracellular signalling. Disturbance of this regulation results in a variety of different diseases including epilepsy, heart failure, cystic fibrosis and diabetes. Therefore, these effects should be considered when using CK2 inhibition as a treatment option for cancer.},
}
@article {pmid33082141,
year = {2020},
author = {Pinci, F and Gaidt, MM and Jung, C and Kuut, G and Jackson, MA and Bauernfried, S and Hornung, V},
title = {C-tag TNF: a reporter system to study TNF shedding.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {52},
pages = {18065-18075},
doi = {10.1074/jbc.RA120.015248},
pmid = {33082141},
issn = {1083-351X},
abstract = {TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.},
}
@article {pmid33081350,
year = {2020},
author = {Fernandez-Garcia, L and Pacios, O and González-Bardanca, M and Blasco, L and Bleriot, I and Ambroa, A and López, M and Bou, G and Tomás, M},
title = {Viral Related Tools against SARS-CoV-2.},
journal = {Viruses},
volume = {12},
number = {10},
pages = {},
pmid = {33081350},
issn = {1999-4915},
mesh = {Antibodies, Viral/immunology/therapeutic use ; Antiviral Agents/therapeutic use ; Bacteriophages ; Betacoronavirus/isolation &amp; purification ; COVID-19 ; CRISPR-Cas Systems ; Coronavirus Infections/*therapy/*virology ; Humans ; Pandemics ; Phage Therapy ; Pneumonia, Viral/*therapy/*virology ; SARS-CoV-2 ; Viral Vaccines/immunology ; },
abstract = {At the end of 2019, a new disease appeared and spread all over the world, the COVID-19, produced by the coronavirus SARS-CoV-2. As a consequence of this worldwide health crisis, the scientific community began to redirect their knowledge and resources to fight against it. Here we summarize the recent research on viruses employed as therapy and diagnostic of COVID-19: (i) viral-vector vaccines both in clinical trials and pre-clinical phases; (ii) the use of bacteriophages to find antibodies specific to this virus and some studies of how to use the bacteriophages themselves as a treatment against viral diseases; and finally, (iii) the use of CRISPR-Cas technology both to obtain a fast precise diagnose of the patient and also the possible use of this technology as a cure.},
}
@article {pmid33079628,
year = {2021},
author = {Tyagi, S and Kumar, R and Kumar, V and Won, SY and Shukla, P},
title = {Engineering disease resistant plants through CRISPR-Cas9 technology.},
journal = {GM crops &amp; food},
volume = {12},
number = {1},
pages = {125-144},
pmid = {33079628},
issn = {2164-5701},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/genetics ; Gene Editing ; Plant Diseases/genetics ; },
abstract = {Plants are susceptible to phytopathogens, including bacteria, fungi, and viruses, which cause colossal financial shortfalls (pre- and post-harvest) and threaten global food safety. To combat with these phytopathogens, plant possesses two-layer of defense in the form of PAMP-triggered immunity (PTI), or Effectors-triggered immunity (ETI). The understanding of plant-molecular interactions and revolution of high-throughput molecular techniques have opened the door for innovations in developing pathogen-resistant plants. In this context, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has transformed genome editing (GE) technology and being harnessed for altering the traits. Here we have summarized the complexities of plant immune system and the use of CRISPR-Cas9 to edit the various components of plant immune system to acquire long-lasting resistance in plants against phytopathogens. This review also sheds the light on the limitations of CRISPR-Cas9 system, regulation of CRISPR-Cas9 edited crops and future prospective of this technology.},
}
@article {pmid33078994,
year = {2020},
author = {Lenskaia, T and Boley, D},
title = {Prokaryote autoimmunity in the context of self-targeting by CRISPR-Cas systems.},
journal = {Journal of bioinformatics and computational biology},
volume = {18},
number = {5},
pages = {2050033},
doi = {10.1142/S021972002050033X},
pmid = {33078994},
issn = {1757-6334},
abstract = {Prokaryote adaptive immunity (CRISPR-Cas systems) can be a threat to its carriers. We analyze the risks of autoimmune reactions related to adaptive immunity in prokaryotes by computational methods. We found important differences between bacteria and archaea with respect to autoimmunity potential. According to the results of our analysis, CRISPR-Cas systems in bacteria are more prone to self-targeting even though they possess fewer spacers per organism on average than archaea. The results of our study provide opportunities to use self-targeting in prokaryotes for biological and medical applications.},
}
@article {pmid33077967,
year = {2020},
author = {Csörgő, B and León, LM and Chau-Ly, IJ and Vasquez-Rifo, A and Berry, JD and Mahendra, C and Crawford, ED and Lewis, JD and Bondy-Denomy, J},
title = {A compact Cascade-Cas3 system for targeted genome engineering.},
journal = {Nature methods},
volume = {17},
number = {12},
pages = {1183-1190},
pmid = {33077967},
issn = {1548-7105},
abstract = {CRISPR-Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7-424 kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade-Cas3, but not Cas9. A transferable 'all-in-one' vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR-Cas use was enhanced with an 'anti-anti-CRISPR' strategy. P. aeruginosa Type I-C Cascade-Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.},
}
@article {pmid33077929,
year = {2020},
author = {Pilosof, S and Alcalá-Corona, SA and Wang, T and Kim, T and Maslov, S and Whitaker, R and Pascual, M},
title = {The network structure and eco-evolutionary dynamics of CRISPR-induced immune diversification.},
journal = {Nature ecology &amp; evolution},
volume = {4},
number = {12},
pages = {1650-1660},
pmid = {33077929},
issn = {2397-334X},
support = {CCF C2480//Cystic Fibrosis Foundation (CF Foundation)/ ; },
abstract = {As a heritable sequence-specific adaptive immune system, CRISPR-Cas is a powerful force shaping strain diversity in host-virus systems. While the diversity of CRISPR alleles has been explored, the associated structure and dynamics of host-virus interactions have not. We explore the role of CRISPR in mediating the interplay between host-virus interaction structure and eco-evolutionary dynamics in a computational model and compare the results with three empirical datasets from natural systems. We show that the structure of the networks describing who infects whom and the degree to which strains are immune, are respectively modular (containing groups of hosts and viruses that interact strongly) and weighted-nested (specialist hosts are more susceptible to subsets of viruses that in turn also infect the more generalist hosts with many spacers matching many viruses). The dynamic interplay between these networks influences transitions between dynamical regimes of virus diversification and host control. The three empirical systems exhibit weighted-nested immunity networks, a pattern our theory shows is indicative of hosts able to suppress virus diversification. Previously missing from studies of microbial host-pathogen systems, the immunity network plays a key role in the coevolutionary dynamics.},
}
@article {pmid33076477,
year = {2020},
author = {Jin, S and Bae, J and Song, Y and Pearcy, N and Shin, J and Kang, S and Minton, NP and Soucaille, P and Cho, BK},
title = {Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals.},
journal = {International journal of molecular sciences},
volume = {21},
number = {20},
pages = {},
pmid = {33076477},
issn = {1422-0067},
support = {2018M3D3A1A01055733//The C1 Gas Refinery Program/ ; 2018K1A3A1A21044063//The Korea-France Partnership Program for C1 Refinery/ ; },
abstract = {Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.},
}
@article {pmid32528662,
year = {2020},
author = {Baker, C and Hayden, MS},
title = {Gene editing in dermatology: Harnessing CRISPR for the treatment of cutaneous disease.},
journal = {F1000Research},
volume = {9},
number = {},
pages = {281},
pmid = {32528662},
issn = {2046-1402},
abstract = {The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized gene editing research. Through the repurposing of programmable RNA-guided CRISPR-associated (Cas) nucleases, CRISPR-based genome editing systems allow for the precise modification of specific sites in the human genome and inspire novel approaches for the study and treatment of inherited and acquired human diseases. Here, we review how CRISPR technologies have stimulated key advances in dermatologic research. We discuss the role of CRISPR in genome editing for cutaneous disease and highlight studies on the use of CRISPR-Cas technologies for genodermatoses, cutaneous viruses and bacteria, and melanoma. Additionally, we examine key limitations of current CRISPR technologies, including the challenges these limitations pose for the widespread therapeutic application of CRISPR-based therapeutics.},
}
@article {pmid33074435,
year = {2020},
author = {Tran, MT and Doan, DTH and Kim, J and Song, YJ and Sung, YW and Das, S and Kim, EJ and Son, GH and Kim, SH and Van Vu, T and Kim, JY},
title = {CRISPR/Cas9-based precise excision of SlHyPRP1 domain(s) to obtain salt stress-tolerant tomato.},
journal = {Plant cell reports},
volume = {},
number = {},
pages = {},
pmid = {33074435},
issn = {1432-203X},
support = {2020R1I1A1A01072130//National Research Foundation of Korea (KR)/ ; 2020M3A9I4038352//National Research Foundation of Korea/ ; 2020R1A6A1A03044344//National Research Foundation of Korea/ ; SSAC, Grant PJ01322601//The Next-Generation BioGreen 21 Program/ ; NBT, Grant PJ01478401//The Program for New Plant Breeding Techniques, Rural Development Administration (RDA), Korea/ ; },
abstract = {KEY MESSAGE: CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.},
}
@article {pmid33074412,
year = {2020},
author = {Dhokane, D and Bhadra, B and Dasgupta, S},
title = {CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein.},
journal = {Molecular biology reports},
volume = {47},
number = {11},
pages = {8747-8755},
doi = {10.1007/s11033-020-05922-5},
pmid = {33074412},
issn = {1573-4978},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) - Cas associated protein 9 (Cas9) system is very precise, efficient and relatively simple in creating genetic modifications at a predetermined locus in the genome. Genome editing with Cas9 ribonucleoproteins (RNPs) has reduced cytotoxic effects, off-target cleavage and increased on-target activity and the editing efficiencies. The unicellular alga Chlamydomonas reinhardtii is an emerging model for studying the production of high-value products for industrial applications. Development of C. reinhardtii as an industrial biotechnology host can be achieved more efficiently through genetic modifications using genome editing tools. We made an attempt to target MAA7 gene that encodes the tryptophan synthase β-Subunit using CRISPR-Cas9 RNPs to demonstrate knock-out and knock-in through homology-dependent repair template at the target site. In this study, we have demonstrated targeted gene knock-out in C. reinhardtii using programmed RNPs. Targeted editing of MAA7 gene was confirmed by sequencing the clones that were resistant to 5-Fluoroindole (5-FI). Non-homologous end joining (NHEJ) repair mechanism led to insertion, deletion, and/or base substitution in the Cas9 cleavage vicinity, encoding non-functional MAA7 protein product (knock-out), conferring resistance to 5-FI. Here, we report an efficient protocol for developing knock-out mutants in Chlamydomonas using CRISPR-Cas9 RNPs. The high potential efficiency of editing may also eliminate the need to select mutants by phenotype. These research findings would be more likely applied to other green algae for developing green cell factories to produce high-value molecules.},
}
@article {pmid33074086,
year = {2020},
author = {Hampton, HG and Smith, LM and Ferguson, S and Meaden, S and Jackson, SA and Fineran, PC},
title = {Functional genomics reveals the toxin-antitoxin repertoire and AbiE activity in Serratia.},
journal = {Microbial genomics},
volume = {6},
number = {11},
pages = {},
pmid = {33074086},
issn = {2057-5858},
abstract = {Bacteriophage defences are divided into innate and adaptive systems. Serratia sp. ATCC 39006 has three CRISPR-Cas adaptive immune systems, but its innate immune repertoire is unknown. Here, we re-sequenced and annotated the Serratia genome and predicted its toxin-antitoxin (TA) systems. TA systems can provide innate phage defence through abortive infection by causing infected cells to 'shut down', limiting phage propagation. To assess TA system function on a genome-wide scale, we utilized transposon insertion and RNA sequencing. Of the 32 TA systems predicted bioinformatically, 4 resembled pseudogenes and 11 were demonstrated to be functional based on transposon mutagenesis. Three functional systems belonged to the poorly characterized but widespread, AbiE, abortive infection/TA family. AbiE is a type IV TA system with a predicted nucleotidyltransferase toxin. To investigate the mode of action of this toxin, we measured the transcriptional response to AbiEii expression. We observed dysregulated levels of tRNAs and propose that the toxin targets tRNAs resulting in bacteriostasis. A recent report on a related toxin shows this occurs through addition of nucleotides to tRNA(s). This study has demonstrated the utility of functional genomics for probing TA function in a high-throughput manner, defined the TA repertoire in Serratia and shown the consequences of AbiE induction.},
}
@article {pmid33072060,
year = {2020},
author = {Zhang, Z and Li, Y and Luo, L and Hao, J and Li, J},
title = {Corrigendum: Characterization of cmcp Gene as a Pathogenicity Factor of Ceratocystis manginecans.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {595238},
doi = {10.3389/fmicb.2020.595238},
pmid = {33072060},
issn = {1664-302X},
abstract = {[This corrects the article DOI: 10.3389/fmicb.2020.01824.].},
}
@article {pmid33072011,
year = {2020},
author = {Yimer, SA and Kalayou, S and Homberset, H and Birhanu, AG and Riaz, T and Zegeye, ED and Lutter, T and Abebe, M and Holm-Hansen, C and Aseffa, A and Tønjum, T},
title = {Lineage-Specific Proteomic Signatures in the Mycobacterium tuberculosis Complex Reveal Differential Abundance of Proteins Involved in Virulence, DNA Repair, CRISPR-Cas, Bioenergetics and Lipid Metabolism.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {550760},
pmid = {33072011},
issn = {1664-302X},
abstract = {Despite the discovery of the tubercle bacillus more than 130 years ago, its physiology and the mechanisms of virulence are still not fully understood. A comprehensive analysis of the proteomes of members of the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages 3, 4, 5, and 7 was conducted to better understand the evolution of virulence and other physiological characteristics. Unique and shared proteomic signatures in these modern, pre-modern and ancient MTBC lineages, as deduced from quantitative bioinformatics analyses of high-resolution mass spectrometry data, were delineated. The main proteomic findings were verified by using immunoblotting. In addition, analysis of multiple genome alignment of members of the same lineages was performed. Label-free peptide quantification of whole cells from MTBC lineages 3, 4, 5, and 7 yielded a total of 38,346 unique peptides derived from 3092 proteins, representing 77% coverage of the predicted proteome. MTBC lineage-specific differential expression was observed for 539 proteins. Lineage 7 exhibited a markedly reduced abundance of proteins involved in DNA repair, type VII ESX-3 and ESX-1 secretion systems, lipid metabolism and inorganic phosphate uptake, and an increased abundance of proteins involved in alternative pathways of the TCA cycle and the CRISPR-Cas system as compared to the other lineages. Lineages 3 and 4 exhibited a higher abundance of proteins involved in virulence, DNA repair, drug resistance and other metabolic pathways. The high throughput analysis of the MTBC proteome by super-resolution mass spectrometry provided an insight into the differential expression of proteins between MTBC lineages 3, 4, 5, and 7 that may explain the slow growth and reduced virulence, metabolic flexibility, and the ability to survive under adverse growth conditions of lineage 7.},
}
@article {pmid33069188,
year = {2020},
author = {Hameed, A and Hussain, SA and Ijaz, MU and Umer, M},
title = {Deletions of the Idh1, Eco1, Rom2, and Taf10 Genes Differently Control the Hyphal Growth, Drug Tolerance, and Virulence of Candida albicans.},
journal = {Folia biologica},
volume = {66},
number = {3},
pages = {91-103},
pmid = {33069188},
issn = {0015-5500},
mesh = {Acetyltransferases/deficiency/genetics/physiology ; Antifungal Agents/pharmacology ; CRISPR-Cas Systems ; Calcium/physiology ; Candida albicans/drug effects/genetics/pathogenicity/*physiology ; Cations/pharmacology ; Cell Adhesion ; Cell Cycle ; Cell Wall/drug effects ; Chitinases/pharmacology ; DNA Damage ; Fungal Proteins/genetics/*physiology ; Gene Deletion ; *Genes, Fungal ; Glucan Endo-1,3-beta-D-Glucosidase/pharmacology ; Hyphae/growth &amp; development ; Isocitrate Dehydrogenase/deficiency/genetics/physiology ; Open Reading Frames ; Reproduction, Asexual ; TATA-Binding Protein Associated Factors/deficiency/genetics/physiology ; Virulence/genetics ; },
abstract = {The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.},
}
@article {pmid33068435,
year = {2020},
author = {Huang, L and Yang, B and Yi, H and Asif, A and Wang, J and Lithgow, T and Zhang, H and Minhas, FUAA and Yin, Y},
title = {AcrDB: a database of anti-CRISPR operons in prokaryotes and viruses.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa857},
pmid = {33068435},
issn = {1362-4962},
abstract = {CRISPR-Cas is an anti-viral mechanism of prokaryotes that has been widely adopted for genome editing. To make CRISPR-Cas genome editing more controllable and safer to use, anti-CRISPR proteins have been recently exploited to prevent excessive/prolonged Cas nuclease cleavage. Anti-CRISPR (Acr) proteins are encoded by (pro)phages/(pro)viruses, and have the ability to inhibit their host's CRISPR-Cas systems. We have built an online database AcrDB (http://bcb.unl.edu/AcrDB) by scanning ∼19 000 genomes of prokaryotes and viruses with AcrFinder, a recently developed Acr-Aca (Acr-associated regulator) operon prediction program. Proteins in Acr-Aca operons were further processed by two machine learning-based programs (AcRanker and PaCRISPR) to obtain numerical scores/ranks. Compared to other anti-CRISPR databases, AcrDB has the following unique features: (i) It is a genome-scale database with the largest collection of data (39 799 Acr-Aca operons containing Aca or Acr homologs); (ii) It offers a user-friendly web interface with various functions for browsing, graphically viewing, searching, and batch downloading Acr-Aca operons; (iii) It focuses on the genomic context of Acr and Aca candidates instead of individual Acr protein family and (iv) It collects data with three independent programs each having a unique data mining algorithm for cross validation. AcrDB will be a valuable resource to the anti-CRISPR research community.},
}
@article {pmid33068408,
year = {2020},
author = {Eki, R and She, J and Parlak, M and Benamar, M and Du, KP and Kumar, P and Abbas, T},
title = {A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair.},
journal = {Nucleic acids research},
volume = {48},
number = {21},
pages = {e126},
doi = {10.1093/nar/gkaa897},
pmid = {33068408},
issn = {1362-4962},
support = {R01 GM135376/GM/NIGMS NIH HHS/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; },
mesh = {Ataxia Telangiectasia Mutated Proteins/metabolism ; Biological Assay/*methods ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; DNA Ligase ATP/metabolism ; *DNA Repair ; DNA Repair Enzymes/metabolism ; DNA-Binding Proteins/metabolism ; Fluorescent Dyes/*chemistry ; *Genes, Reporter ; HEK293 Cells ; Humans ; },
abstract = {DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.},
}
@article {pmid33068118,
year = {2020},
author = {Chan, AN and Wang, LL and Zhu, YJ and Fan, YY and Zhuang, JY and Zhang, ZH},
title = {Identification through fine mapping and verification using CRISPR/Cas9-targeted mutagenesis for a minor QTL controlling grain weight in rice.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00122-020-03699-6},
pmid = {33068118},
issn = {1432-2242},
support = {31701072//National Natural Science Foundation of China/ ; LQ17C130004//Zhejiang Provincial Natural Science Foundation of China/ ; LQ18C130002//Zhejiang Provincial Natural Science Foundation of China/ ; 3240303476//Organization for Women in Science for the Developing World and Swedish International Development Cooperation Agency/ ; },
abstract = {KEY MESSAGE: A minor QTL for grain weight in rice, qTGW1.2b, was fine-mapped. Its casual gene OsVQ4 was confirmed through CRISPR/Cas9-targeted mutagenesis, exhibiting an effect that was larger than the original QTL effect. The CRISPR/Cas system exhibits a great potential for rice improvement, but the application was severely hindered due to insufficient target genes, especial the lack of validated genes underlying quantitative trait loci having small effects. In this study, a minor QTL for grain weight, qTGW1.2b, was fine-mapped into a 44.0 kb region using seven sets of near isogenic lines (NILs) developed from the indica rice cross (Zhenshan 97)3/Milyang 46, followed by validation of the causal gene using CRISPR/Cas9-targeted mutagenesis. In the NIL populations, 1000-grain weight of the Zhenshan 97 homozygous lines decreased by 0.9-2.0% compared with the Milyang 46 homozygous lines. A gene encoding VQ-motif protein, OsVQ4, was identified as the candidate gene based on parental sequence differences. The effect of OsVQ4 was confirmed by creating CRISPR/Cas9 knockout lines, whose 1000-grain weight decreased by 2.8-9.8% compared with the wild-type transgenic line and the recipient. These results indicate that applying genome editing system could create novel alleles with large phenotypic variation at minor QTLs, which is an effective way to validate causal genes of minor QTLs. Our study establishes a strategy for cloning minor QTLs, which could also be used to identify a large number of potential target genes for the application of CRISPR/Cas system.},
}
@article {pmid33067443,
year = {2020},
author = {Huang, X and Sun, W and Cheng, Z and Chen, M and Li, X and Wang, J and Sheng, G and Gong, W and Wang, Y},
title = {Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5241},
pmid = {33067443},
issn = {2041-1723},
mesh = {Catalysis ; Catalytic Domain ; DNA Cleavage ; DNA, Single-Stranded/chemistry/genetics/*metabolism ; Deoxyribonuclease I/*chemistry/genetics/*metabolism ; Ions/chemistry/metabolism ; Metals/chemistry/*metabolism ; },
abstract = {To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2-crRNA-DNA complexes and a Cas12i2-crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1-8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9-15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.},
}
@article {pmid33066557,
year = {2020},
author = {Saha, C and Horst-Kreft, D and Kross, I and van der Spek, PJ and Louwen, R and van Baarlen, P},
title = {Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {33066557},
issn = {2073-4425},
abstract = {The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.},
}
@article {pmid33060338,
year = {2020},
author = {Cohen, J},
title = {A cut above: pair that developed CRISPR earns historic award.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6514},
pages = {271-272},
doi = {10.1126/science.370.6514.271},
pmid = {33060338},
issn = {1095-9203},
mesh = {Animals ; *CRISPR-Cas Systems ; Chemistry/*history ; Embryo Research/ethics/history ; France ; Gene Editing/*history ; History, 21st Century ; Humans ; *Nobel Prize ; Patents as Topic/history ; United States ; },
}
@article {pmid33059029,
year = {2020},
author = {Kondrateva, E and Demchenko, A and Lavrov, A and Smirnikhina, S},
title = {An overview of currently available molecular Cas-tools for precise genome modification.},
journal = {Gene},
volume = {},
number = {},
pages = {145225},
doi = {10.1016/j.gene.2020.145225},
pmid = {33059029},
issn = {1879-0038},
abstract = {CRISPR-Cas system was first mentioned in 1987, and over the years have been studied so active that now it becomes the state-of-the-art tool for genome editing. Its working principle is based on Cas nuclease ability to bind short RNA, which targets it to complementary DNA or RNA sequence for highly precise cleavage. This alone or together with donor DNA allows to modify targeted sequence in different ways. Considering the many limitations of using native CRISPR-Cas systems, scientists around the world are working on creating modified variants to improve their specificity and efficiency in different objects. In addition, the use of the Cas effectors' targeting function in complex systems with other proteins is a promising work direction, as a result of which new tools are created with features such as single base editing, editing DNA without break and donor DNA, activation and repression of transcription, epigenetic regulation, modifying of different repair pathways involvement etc. In this review, we decided to consider in detail exactly this issue of variants of Cas effectors, their modifications and fusion molecules, which improve DNA-targeting and expand the scope of Cas effectors.},
}
@article {pmid33057400,
year = {2020},
author = {Maroc, L and Zhou-Li, Y and Boisnard, S and Fairhead, C},
title = {A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata.},
journal = {PLoS genetics},
volume = {16},
number = {10},
pages = {e1008627},
pmid = {33057400},
issn = {1553-7404},
abstract = {Mating-type switching is a complex mechanism that promotes sexual reproduction in Saccharomycotina. In the model species Saccharomyces cerevisiae, mating-type switching is initiated by the Ho endonuclease that performs a site-specific double-strand break (DSB) at MAT, repaired by homologous recombination (HR) using one of the two silent mating-type loci, HMLalpha and HMRa. The reasons why all the elements of the mating-type switching system have been conserved in some Saccharomycotina, that do not show a sexual cycle nor mating-type switching, remain unknown. To gain insight on this phenomenon, we used the yeast Candida glabrata, phylogenetically close to S. cerevisiae, and for which no spontaneous and efficient mating-type switching has been observed. We have previously shown that expression of S. cerevisiae's Ho (ScHo) gene triggers mating-type switching in C. glabrata, but this leads to massive cell death. In addition, we unexpectedly found, that not only MAT but also HML was cut in this species, suggesting the formation of multiple chromosomal DSBs upon HO induction. We now report that HMR is also cut by ScHo in wild-type strains of C. glabrata. To understand the link between mating-type switching and cell death in C. glabrata, we constructed strains mutated precisely at the Ho recognition sites. We find that even when HML and HMR are protected from the Ho-cut, introducing a DSB at MAT is sufficient to induce cell death, whereas one DSB at HML or HMR is not. We demonstrate that mating-type switching in C. glabrata can be triggered using CRISPR-Cas9, without high lethality. We also show that switching is Rad51-dependent, as in S. cerevisiae, but that donor preference is not conserved in C. glabrata. Altogether, these results suggest that a DSB at MAT can be repaired by HR in C. glabrata, but that repair is prevented by ScHo.},
}
@article {pmid33057349,
year = {2020},
author = {Gallagher, DN and Pham, N and Tsai, AM and Janto, AN and Choi, J and Ira, G and Haber, JE},
title = {A Rad51-independent pathway promotes single-strand template repair in gene editing.},
journal = {PLoS genetics},
volume = {16},
number = {10},
pages = {e1008689},
pmid = {33057349},
issn = {1553-7404},
support = {R01 GM125650/GM/NIGMS NIH HHS/United States ; R35 GM127029/GM/NIGMS NIH HHS/United States ; T32 GM007122/GM/NIGMS NIH HHS/United States ; },
abstract = {The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.},
}
@article {pmid33057331,
year = {2020},
author = {Liu, J and Zuo, Z and Zou, M and Finkel, T and Liu, S},
title = {Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen.},
journal = {PLoS genetics},
volume = {16},
number = {10},
pages = {e1009068},
pmid = {33057331},
issn = {1553-7404},
support = {R01 AI145879/AI/NIAID NIH HHS/United States ; R56 AI148134/AI/NIAID NIH HHS/United States ; },
abstract = {Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.},
}
@article {pmid33057066,
year = {2020},
author = {Zhao, C and Liu, H and Xiao, T and Wang, Z and Nie, X and Li, X and Qian, P and Qin, L and Han, X and Zhang, J and Ruan, J and Zhu, M and Miao, YL and Zuo, B and Yang, K and Xie, S and Zhao, S},
title = {CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5178},
pmid = {33057066},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calreticulin/genetics/metabolism ; Encephalitis Virus, Japanese/metabolism/*pathogenicity ; Encephalitis, Japanese/*pathology/virology ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Gene Library ; HEK293 Cells ; Host-Pathogen Interactions/*genetics ; Humans ; Membrane Proteins/genetics/metabolism ; RNA, Guide/genetics ; RNA, Small Interfering/metabolism ; Sus scrofa ; *Virus Replication ; },
abstract = {Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.},
}
@article {pmid33052901,
year = {2020},
author = {Semesta, KM and Tian, R and Kampmann, M and von Zastrow, M and Tsvetanova, NG},
title = {A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.},
journal = {PLoS genetics},
volume = {16},
number = {10},
pages = {e1009103},
pmid = {33052901},
issn = {1553-7404},
support = {R00 MH109633/MH/NIMH NIH HHS/United States ; R01 DA010711/DA/NIDA NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; },
abstract = {G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.},
}
@article {pmid33049228,
year = {2020},
author = {Niu, Y and Yang, L and Gao, T and Dong, C and Zhang, B and Yin, P and Hopp, AK and Li, D and Gan, R and Wang, H and Liu, X and Cao, X and Xie, Y and Meng, X and Deng, H and Zhang, X and Ren, J and Hottiger, MO and Chen, Z and Zhang, Y and Liu, X and Feng, Y},
title = {A Type I-F Anti-CRISPR Protein Inhibits the CRISPR-Cas Surveillance Complex by ADP-Ribosylation.},
journal = {Molecular cell},
volume = {80},
number = {3},
pages = {512-524.e5},
doi = {10.1016/j.molcel.2020.09.015},
pmid = {33049228},
issn = {1097-4164},
abstract = {CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems.},
}
@article {pmid33048924,
year = {2020},
author = {Mutalik, VK and Adler, BA and Rishi, HS and Piya, D and Zhong, C and Koskella, B and Kutter, EM and Calendar, R and Novichkov, PS and Price, MN and Deutschbauer, AM and Arkin, AP},
title = {High-throughput mapping of the phage resistance landscape in E. coli.},
journal = {PLoS biology},
volume = {18},
number = {10},
pages = {e3000877},
pmid = {33048924},
issn = {1545-7885},
mesh = {Bacterial Proteins/genetics/metabolism ; Bacteriophages/drug effects/*physiology ; Biosynthetic Pathways/drug effects ; CRISPR-Cas Systems/genetics ; Cyclic GMP/analogs &amp; derivatives/pharmacology ; DNA/genetics ; Down-Regulation/drug effects/genetics ; Escherichia coli/drug effects/genetics/*virology ; Gene Expression Regulation, Bacterial/drug effects ; Genes, Essential ; Genome, Bacterial ; Mutation/genetics ; Phenotype ; Reproducibility of Results ; Suppression, Genetic ; },
abstract = {Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.},
}
@article {pmid33047464,
year = {2020},
author = {Van Vu, T and Thi Hai Doan, D and Kim, J and Sung, YW and Thi Tran, M and Song, YJ and Das, S and Kim, JY},
title = {CRISPR/Cas-based precision genome editing via microhomology-mediated end joining.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13490},
pmid = {33047464},
issn = {1467-7652},
support = {2020M3A9I4038352//National Research Foundation of Korea/ ; 2020R1A6A1A03044344//National Research Foundation of Korea/ ; 2020R1I1A1A01072130//National Research Foundation of Korea/ ; PJ01478401//The Program for New Plant Breeding Techniques, Rural Development Administration, Republic of Korea/ ; PJ01322601//Next-Generation BioGreen 21 Program, Republic of Korea/ ; },
abstract = {Gene editing and/or allele introgression with absolute precision and control appear to be the ultimate goals of genetic engineering. Precision genome editing in plants has been developed through various approaches, including oligonucleotide-directed mutagenesis (ODM), base editing, prime editing and especially homologous recombination (HR)-based gene targeting. With the advent of CRISPR/Cas for the targeted generation of DNA breaks (single-stranded breaks (SSBs) or double-stranded breaks (DSBs)), a substantial advancement in HR-mediated precise editing frequencies has been achieved. Nonetheless, further research needs to be performed for commercially viable applications of precise genome editing; hence, an alternative innovative method for genome editing may be required. Within this scope, we summarize recent progress regarding precision genome editing mediated by microhomology-mediated end joining (MMEJ) and discuss their potential applications in crop improvement.},
}
@article {pmid33046904,
year = {2020},
author = {Zhu, H and Li, C and Gao, C},
title = {Publisher Correction: Applications of CRISPR-Cas in agriculture and plant biotechnology.},
journal = {Nature reviews. Molecular cell biology},
volume = {21},
number = {11},
pages = {712},
doi = {10.1038/s41580-020-00304-y},
pmid = {33046904},
issn = {1471-0080},
abstract = {An amendment to this paper has been published and can be accessed via a link at the top of the paper.},
}
@article {pmid33043922,
year = {2020},
author = {Tian, W and Liu, X and Wang, G and Liu, C},
title = {A hyperbranched transcription-activated CRISPR-Cas12a signal amplification strategy for sensitive microRNA sensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {56},
number = {87},
pages = {13445-13448},
doi = {10.1039/d0cc06034h},
pmid = {33043922},
issn = {1364-548X},
abstract = {A CRISPR-Cas12a-based strategy is developed for sensitive microRNA sensing, in which hyperbranched rolling circle amplification and transcription are integrated for the activation of the trans-cleavage ability of Cas12a. The integrated triplex signal amplification endows the strategy with a low background and high sensitivity. This design expands the scope of the CRISPR-Cas-based sensing toolbox and shows great potential in biological and biomedical studies.},
}
@article {pmid33042266,
year = {2020},
author = {Sun, NH and Chen, DY and Ye, LP and Sheng, G and Gong, JJ and Chen, BH and Lu, YM and Han, F},
title = {CRISPR-Sunspot: Imaging of endogenous low-abundance RNA at the single-molecule level in live cells.},
journal = {Theranostics},
volume = {10},
number = {24},
pages = {10993-11012},
pmid = {33042266},
issn = {1838-7640},
abstract = {CRISPR/Cas-based mRNA imaging has been developed to labeling of high-abundance mRNAs. A lack of non-genetically encoded mRNA-tagged imaging tools has limited our ability to explore the functional distributions of endogenous low-abundance mRNAs in cells. Here, we developed a CRISPR-Sunspot method based on the SunTag signal amplification system that allows efficient imaging of low-abundance mRNAs with CRISPR/Cas9. Methods: We created a stable TRE3G-dCas9-EGFP cell line and generated an Inducible dCas9-EGFP imaging system for assessment of two factors, sgRNA and dCas9, which influence imaging quality. Based on SunTag system, we established a CRISPR-Sunspot imaging system for amplifying signals from single-molecule mRNA in live cells. CRISPR-Sunspot was used to track co-localization of Camk2a mRNA with regulatory protein Xlr3b in neurons. CRISPR-Sunspot combined with CRISPRa was used to determine elevated mRNA molecules. Results: Our results showed that manipulating the expression of fluorescent proteins and sgRNA increased the efficiency of RNA imaging in cells. CRISPR-Sunspot could target endogenous mRNAs in the cytoplasm and amplified signals from single-molecule mRNA. Furthermore, CRISPR-Sunspot was also applied to visualize mRNA distributions with its regulating proteins in neurons. CRISPR-Sunspot detected the co-localization of Camk2a mRNA with overexpressed Xlr3b proteins in the neuronal dendrites. Moreover, we also manipulated CRISPR-Sunspot to detect transcriptional activation of target gene such as HBG1 in live cells. Conclusion: Our findings suggest that CRISPR-Sunspot is a novel applicable imaging tool for visualizing the distributions of low-abundance mRNAs in cells. This study provides a novel strategy to unravel the molecular mechanisms of diseases caused by aberrant mRNA molecules.},
}
@article {pmid33039910,
year = {2021},
author = {Bonini, A and Poma, N and Vivaldi, F and Kirchhain, A and Salvo, P and Bottai, D and Tavanti, A and Di Francesco, F},
title = {Advances in biosensing: The CRISPR/Cas system as a new powerful tool for the detection of nucleic acids.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {192},
number = {},
pages = {113645},
doi = {10.1016/j.jpba.2020.113645},
pmid = {33039910},
issn = {1873-264X},
abstract = {A main challenge in the development of biosensing devices for the identification and quantification of nucleic acids is to avoid the amplification of the genetic material from the sample by polymerase chain reaction (PCR), which is at present necessary to enhance sensitivity and selectivity of assays. PCR has undoubtedly revolutionized genetic analyses, but it requires careful purification procedures that are not easily implemented in point of care (POC) devices. In recent years, a new strategy for nucleic acid detection based on clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein systems (Cas) seems to offer unprecedented possibilities. The coupling of the CRISPR/Cas system with recent isothermal amplification methods is fostering the development of innovative optical and electrochemical POC devices. In this review, the mechanisms of action of several new CRISRP/Cas systems are reported together with their use in biosensing of nucleic acids.},
}
@article {pmid33039728,
year = {2020},
author = {Mahdizadeh, S and Sansom, FM and Lee, SW and Browning, GF and Marenda, MS},
title = {Targeted mutagenesis of Mycoplasma gallisepticum using its endogenous CRISPR/Cas system.},
journal = {Veterinary microbiology},
volume = {250},
number = {},
pages = {108868},
doi = {10.1016/j.vetmic.2020.108868},
pmid = {33039728},
issn = {1873-2542},
abstract = {New, more efficient methods are needed to facilitate studies of gene function in the mycoplasmas. CRISPR/Cas systems, which provide bacteria with acquired immunity against invading nucleic acids, have been developed as tools for genomic editing in a wide range of organisms. We explored the potential for using the endogenous Mycoplasma gallisepticum CRISPR/Cas system to introduce targeted mutations into the chromosome of this important animal pathogen. Three constructs carrying different CRISPR arrays targeting regions in the ksgA gene (pK1-CRISPR, pK-CRISPR-1 and pK-CRISPR-2) were assembled and introduced into M. gallisepticum on an oriC plasmid. The loss of KsgA prevents ribosomal methylation, which in turn confers resistance to the aminoglycoside antimicrobial kasugamycin, enabling selection for ksgA mutants. Analyses of the complete sequence of the ksgA gene in 78 resistant transformants revealed various modifications of the target region, presumably caused by the directed CRISPR/Cas activity of M. gallisepticum. The analyses suggested that M. gallisepticum may utilize a non-homologous end joining (NHEJ) repair system, which can result in deletion or duplication of a short DNA segment in the presence of double-stranded breaks. This study has generated an improved understanding of the M. gallisepticum CRISPR/Cas system, and may also facilitate further development of tools to genetically modify this important pathogen.},
}
@article {pmid33034197,
year = {2020},
author = {Wang, Z and Sui, Y and Li, J and Tian, X and Wang, Q},
title = {Biological control of postharvest fungal decays in citrus: a review.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-10},
doi = {10.1080/10408398.2020.1829542},
pmid = {33034197},
issn = {1549-7852},
abstract = {Citrus (Citrus spp.) species produce a variety of fruits that are popular worldwide. Citrus fruits, however, are susceptible to postharvest decays caused by various pathogenic fungi, including Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii, Aspergillus niger, and Aspergillus flavus. Decays resulting from infections by these pathogens cause a significant reduction in citrus quality and marketable yield. Biological control of postharvest decay utilizing antagonistic bacteria and fungi has been explored as a promising alternative to synthetic fungicides. In the present article, the isolation of antagonists utilized to manage postharvest decays in citrus is reviewed, and the mechanism of action including recent molecular and genomic studies is discussed as well. Several recently-postulated mechanisms of action, such as biofilm formation and an oxidative burst of reactive oxygen species have been highlighted. Improvements in biocontrol efficacy of antagonists through the use of a combination of microbial antagonists and additives are also reviewed. Biological control utilizing bacterial and yeast antagonists is a critical component of an integrated management approach for the sustainable development of the citrus industry. Further research will be needed, however, to explore and utilize beneficial microbial consortia and novel approaches like CRISPR/Cas technology for management of postharvest decays.},
}
@article {pmid33033974,
year = {2020},
author = {Yan, M and Li, J},
title = {Combined application of CRISPR-Cas and stem cells for clinical and basic research.},
journal = {Cell regeneration (London, England)},
volume = {9},
number = {1},
pages = {19},
pmid = {33033974},
issn = {2045-9769},
}
@article {pmid33033246,
year = {2020},
author = {Martinez-Lage, M and Torres-Ruiz, R and Puig-Serra, P and Moreno-Gaona, P and Martin, MC and Moya, FJ and Quintana-Bustamante, O and Garcia-Silva, S and Carcaboso, AM and Petazzi, P and Bueno, C and Mora, J and Peinado, H and Segovia, JC and Menendez, P and Rodriguez-Perales, S},
title = {In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5060},
pmid = {33033246},
issn = {2041-1723},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Doxorubicin/therapeutic use ; Fusion Proteins, bcr-abl/genetics ; Gene Deletion ; Genetic Loci ; Genomic Instability ; HEK293 Cells ; Humans ; Introns/genetics ; Mice, Nude ; Neoplasms/drug therapy/*genetics/pathology ; Oncogene Fusion/*genetics ; Oncogene Proteins, Fusion/genetics ; RNA, Guide/metabolism ; Reproducibility of Results ; Xenograft Model Antitumor Assays ; },
abstract = {Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells.},
}
@article {pmid33033131,
year = {2020},
author = {Sieber, RN and Overballe-Petersen, S and Kaya, H and Larsen, AR and Petersen, A},
title = {Complete Genome Sequences of Methicillin-Resistant Staphylococcus aureus Strains 110900 and 128254, Two Representatives of the CRISPR-Cas-Carrying Sequence Type 630/spa Type t4549 Lineage.},
journal = {Microbiology resource announcements},
volume = {9},
number = {41},
pages = {},
pmid = {33033131},
issn = {2576-098X},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) and spa type t4549 is an emerging lineage in Nordic countries, and some representatives carry the CRISPR-Cas system. Here, the complete genome sequences of two isolates from this lineage are presented, comprising chromosomes of 2,918,239 and 2,877,083 nucleotides, respectively, and a 2,473-nucleotide plasmid carrying erm(C).},
}
@article {pmid33031826,
year = {2020},
author = {Hazafa, A and Mumtaz, M and Farooq, MF and Bilal, S and Chaudhry, SN and Firdous, M and Naeem, H and Ullah, MO and Yameen, M and Mukhtiar, MS and Zafar, F},
title = {CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions.},
journal = {Life sciences},
volume = {263},
number = {},
pages = {118525},
doi = {10.1016/j.lfs.2020.118525},
pmid = {33031826},
issn = {1879-0631},
abstract = {Cancer is one of the most leading causes of death and a major public health problem, universally. According to accumulated data, annually, approximately 8.5 million people died because of the lethality of cancer. Recently, a novel RNA domain-containing endonuclease-based genome engineering technology, namely the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9) have been proved as a powerful technique in the treatment of cancer cells due to its multifunctional properties including high specificity, accuracy, time reducing and cost-effective strategies with minimum off-target effects. The present review investigates the overview of recent studies on the newly developed genome-editing strategy, CRISPR/Cas9, as an excellent pre-clinical therapeutic option in the reduction and identification of new tumor target genes in the solid tumors. Based on accumulated data, we revealed that CRISPR/Cas9 significantly inhibited the robust tumor cell growth (breast, lung, liver, colorectal, and prostate) by targeting the oncogenes, tumor-suppressive genes, genes associated to therapies by inhibitors, genes associated to chemotherapies drug resistance, and suggested that CRISPR/Cas9 could be a potential therapeutic target in inhibiting the tumor cell growth by suppressing the cell-proliferation, metastasis, invasion and inducing the apoptosis during the treatment of malignancies in the near future. The present review also discussed the current challenges and barriers, and proposed future recommendations for a better understanding.},
}
@article {pmid33028993,
year = {2020},
author = {Ledford, H and Callaway, E},
title = {Pioneers of revolutionary CRISPR gene editing win chemistry Nobel.},
journal = {Nature},
volume = {586},
number = {7829},
pages = {346-347},
doi = {10.1038/d41586-020-02765-9},
pmid = {33028993},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Deoxyribonuclease I ; *Gene Editing ; RNA ; },
}
@article {pmid33028827,
year = {2020},
author = {Carlson-Stevermer, J and Kelso, R and Kadina, A and Joshi, S and Rossi, N and Walker, J and Stoner, R and Maures, T},
title = {CRISPRoff enables spatio-temporal control of CRISPR editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {5041},
pmid = {33028827},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*genetics/radiation effects ; Cell Line, Tumor ; DNA Breaks, Double-Stranded ; Feasibility Studies ; Gene Editing/*methods ; HEK293 Cells ; Humans ; *Light ; RNA Stability/*radiation effects ; RNA, Guide/*metabolism/radiation effects ; Ribonucleoproteins/metabolism ; Translocation, Genetic ; },
abstract = {Following introduction of CRISPR-Cas9 components into a cell, genome editing occurs unabated until degradation of its component nucleic acids and proteins by cellular processes. This uncontrolled reaction can lead to unintended consequences including off-target editing and chromosomal translocations. To address this, we develop a method for light-induced degradation of sgRNA termed CRISPRoff. Here we show that light-induced inactivation of ribonucleoprotein attenuates genome editing within cells and allows for titratable levels of editing efficiency and spatial patterning via selective illumination.},
}
@article {pmid33028045,
year = {2020},
author = {Kostyushev, D and Kostyusheva, A and Brezgin, S and Smirnov, V and Volchkova, E and Lukashev, A and Chulanov, V},
title = {Gene Editing by Extracellular Vesicles.},
journal = {International journal of molecular sciences},
volume = {21},
number = {19},
pages = {},
pmid = {33028045},
issn = {1422-0067},
support = {20-15-00373//Russian Science Foundation/ ; },
abstract = {CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.},
}
@article {pmid33027946,
year = {2020},
author = {Shalaby, K and Aouida, M and El-Agnaf, O},
title = {Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors.},
journal = {International journal of molecular sciences},
volume = {21},
number = {19},
pages = {},
pmid = {33027946},
issn = {1422-0067},
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes.},
}
@article {pmid33023639,
year = {2020},
author = {Jiang, YY and Chai, YP and Lu, MH and Han, XL and Lin, Q and Zhang, Y and Zhang, Q and Zhou, Y and Wang, XC and Gao, C and Chen, QJ},
title = {Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {257},
pmid = {33023639},
issn = {1474-760X},
abstract = {Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.},
}
@article {pmid33022264,
year = {2020},
author = {Dimitriu, T and Szczelkun, MD and Westra, ER},
title = {Evolutionary Ecology and Interplay of Prokaryotic Innate and Adaptive Immune Systems.},
journal = {Current biology : CB},
volume = {30},
number = {19},
pages = {R1189-R1202},
pmid = {33022264},
issn = {1879-0445},
support = {714478/ERC_/European Research Council/International ; 788405/ERC_/European Research Council/International ; BB/L000873/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Like many organisms, bacteria and archaea have both innate and adaptive immune systems to defend against infection by viruses and other parasites. Innate immunity most commonly relies on the endonuclease-mediated cleavage of any incoming DNA that lacks a specific epigenetic modification, through a system known as restriction-modification. CRISPR-Cas-mediated adaptive immunity relies on the insertion of short DNA sequences from parasite genomes into CRISPR arrays on the host genome to provide sequence-specific protection. The discovery of each of these systems has revolutionised our ability to carry out genetic manipulations, and, as a consequence, the enzymes involved have been characterised in exquisite detail. In comparison, much less is known about the importance of these two arms of the defence for the ecology and evolution of prokaryotes and their parasites. Here, we review our current ecological and evolutionary understanding of these systems in isolation, and discuss the need to study how innate and adaptive immune responses are integrated when they coexist in the same cell.},
}
@article {pmid33021880,
year = {2020},
author = {Hahn, M and Scalliet, G},
title = {One cut to change them all: CRISPR/Cas, a groundbreaking tool for genome editing in Botrytis cinerea and other fungal plant pathogens.},
journal = {Phytopathology},
volume = {},
number = {},
pages = {},
doi = {10.1094/PHYTO-09-20-0379-PER},
pmid = {33021880},
issn = {0031-949X},
abstract = {CRISPR/Cas is a genome editing technology that has opened new dimensions in functional biology. In a recent publication, we present a highly efficient CRISPR/Cas technique for Botrytis cinerea, which dramatically increases our options to mutagenize and modify single or multiple genes. In this Perspectives article, we describe the essential features of the method, and demonstrate with several examples how it opens new avenues for unravelling the virulence mechanisms of Botrytis and other plant pathogenic fungi, and to accelerate research for the identification of new antifungal compounds.},
}
@article {pmid33020633,
year = {2020},
author = {Mamidi, S and Healey, A and Huang, P and Grimwood, J and Jenkins, J and Barry, K and Sreedasyam, A and Shu, S and Lovell, JT and Feldman, M and Wu, J and Yu, Y and Chen, C and Johnson, J and Sakakibara, H and Kiba, T and Sakurai, T and Tavares, R and Nusinow, DA and Baxter, I and Schmutz, J and Brutnell, TP and Kellogg, EA},
title = {A genome resource for green millet Setaria viridis enables discovery of agronomically valuable loci.},
journal = {Nature biotechnology},
volume = {38},
number = {10},
pages = {1203-1210},
pmid = {33020633},
issn = {1546-1696},
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; DNA Transposable Elements/genetics ; Domestication ; Edible Grain/genetics ; Gene Editing ; Genome, Plant/*genetics ; Genotype ; Millets/*genetics ; Phenotype ; Phylogeny ; Plant Proteins/*genetics ; Setaria Plant/*genetics ; },
abstract = {Wild and weedy relatives of domesticated crops harbor genetic variants that can advance agricultural biotechnology. Here we provide a genome resource for the wild plant green millet (Setaria viridis), a model species for studies of C4 grasses, and use the resource to probe domestication genes in the close crop relative foxtail millet (Setaria italica). We produced a platinum-quality genome assembly of S. viridis and de novo assemblies for 598 wild accessions and exploited these assemblies to identify loci underlying three traits: response to climate, a 'loss of shattering' trait that permits mechanical harvest and leaf angle, a predictor of yield in many grass crops. With CRISPR-Cas9 genome editing, we validated Less Shattering1 (SvLes1) as a gene whose product controls seed shattering. In S. italica, this gene was rendered nonfunctional by a retrotransposon insertion in the domesticated loss-of-shattering allele SiLes1-TE (transposable element). This resource will enhance the utility of S. viridis for dissection of complex traits and biotechnological improvement of panicoid crops.},
}
@article {pmid33020327,
year = {2020},
author = {Zebell, SG},
title = {Excising the Mystery of Single-Guide RNA Processing.},
journal = {Plant physiology},
volume = {184},
number = {2},
pages = {572-573},
pmid = {33020327},
issn = {1532-2548},
mesh = {*CRISPR-Cas Systems ; Gene Expression ; Nucleic Acid Conformation ; *RNA, Guide ; },
}
@article {pmid33017688,
year = {2020},
author = {Lata, KS and Vaghasia, V and Bhairappanavar, SB and Kumar, S and Ayachit, G and Patel, S and Das, J},
title = {Whole genome sequencing and de novo assembly of three virulent Indian isolates of Leptospira.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {85},
number = {},
pages = {104579},
doi = {10.1016/j.meegid.2020.104579},
pmid = {33017688},
issn = {1567-7257},
abstract = {Leptospirosis is a re-emerging bacterial zoonosis caused by pathogenic Leptospira, with a worldwide distribution and becoming a major public health concern. Prophylaxis of this disease is difficult due to several factors such as non-specific variable clinical manifestation, presence of a large number of serovar, species and asymptomatic reservoir hosts, lack of proper diagnostics and vaccines. Despite its global importance and severity of the disease, knowledge about the molecular mechanism of pathogenesis and evolution of pathogenic species of Leptospira remains limited. In this study, we sequenced and analyzed three highly pathogenic species of Indian isolates of Leptospira (interrogans, santarosai, and kirschneri). Additionally, we identified some virulence-related and CRISPR-Cas genes. The virulent analysis showed 232 potential virulence factors encoding proteins in L. interrogans strain Salinem and L. santarosai strain M-4 genome. While the genome of L. kirschneri strain Wumalasena was predicted to encode 198 virulence factor proteins. The variant calling analysis revealed 1151, 19,786, and 22,996 single nucleotide polymorphisms (SNPs) for L. interrogans strain Salinem, L. kirschneri strain Wumalasena and L. santarosai strain M-4, respectively, with a maximum of 5315 missense and 12,221 synonymous mutations for L. santarosai strain M-4. The structural analyses of genomes indicated potential evidence of inversions and structural rearrangment in all three genomes. The availability of these genome sequences and in silico analysis of Leptospira will provide a basis for a deeper understanding of their molecular diversity and pathogenesis mechanism, and further pave a way towards proper management of the disease.},
}
@article {pmid33014683,
year = {2020},
author = {Meena, MR and Kumar, R and Chinnaswamy, A and Karuppaiyan, R and Kulshreshtha, N and Ram, B},
title = {Current breeding and genomic approaches to enhance the cane and sugar productivity under abiotic stress conditions.},
journal = {3 Biotech},
volume = {10},
number = {10},
pages = {440},
pmid = {33014683},
issn = {2190-572X},
abstract = {Sugarcane (Saccharum spp.) crop is vulnerable to many abiotic stresses such as drought, salinity, waterlogging, cold and high temperature due to climate change. Over the past few decades new breeding and genomic approaches have been used to enhance the genotypic performance under abiotic stress conditions. In sugarcane, introgression of genes from wild species and allied genera for abiotic stress tolerance traits plays a significant role in the development of several stress-tolerant varieties. Moreover, the genomics and transcriptomics approaches have helped to elucidate the key genes/TFs and pathways involved in abiotic stress tolerance in sugarcane. Several novel miRNAs families /proteins or regulatory elements that are responsible for drought, salinity, and cold tolerance have been identified through high-throughput sequencing. The existing sugarcane monoploid genome sequence information opens new gateways and opportunities for researchers to improve the desired traits through efficient genome editing tools, such as the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas) system. TALEN mediated mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane significantly reduces the lignin content in the cell wall which is amenable for biofuel production from lignocellulosic biomass. In this review, we focus on current breeding with genomic approaches and their substantial role in enhancing cane production under the abiotic stress conditions, which is expected to provide new insights to plant breeders and biotechnologists to modify their strategy in developing stress-tolerant sugarcane varieties, which can highlight the future demand of cane, bio-energy, and viability of sugar industries.},
}
@article {pmid33014011,
year = {2020},
author = {Rabinowitz, R and Almog, S and Darnell, R and Offen, D},
title = {CrisPam: SNP-Derived PAM Analysis Tool for Allele-Specific Targeting of Genetic Variants Using CRISPR-Cas Systems.},
journal = {Frontiers in genetics},
volume = {11},
number = {},
pages = {851},
pmid = {33014011},
issn = {1664-8021},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising novel technology that holds the potential of treating genetic diseases. Safety and specificity of the treatment are to be further studied and developed prior to implementation of the technology into the clinic. The guide-RNA (gRNA) allows precise position-specific DNA targeting, although it may tolerate small changes such as point mutations. The permissive nature of the CRISPR-Cas system makes allele-specific targeting a challenging goal. Hence, an allele-specific targeting approach is in need for future treatments of heterozygous patients suffering from diseases caused by dominant negative mutations. The single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach allows highly allele-specific DNA cleavage due to the existence of a novel PAM sequence only at the target allele. Here, we present CrisPam, a computational tool that detects PAMs within the variant allele for allele-specific targeting by CRISPR-Cas systems. The algorithm scans the sequences and attempts to identify the generation of multiple PAMs for a given reference sequence and its variations. A successful result is such that at least a single PAM is generated by the variation nucleotide. Since the PAM is present within the variant allele only, the Cas enzyme will bind the variant allele exclusively. Analyzing a dataset of human pathogenic point mutations revealed that 90% of the analyzed mutations generated at least a single PAM. Thus, the SNP-derived PAM approach is ideal for targeting most of the point mutations in an allele-specific manner. CrisPam simplifies the gRNAs design process to specifically target the allele of interest and scans a wide range of 26 unique PAMs derived from 23 Cas enzymes. CrisPam is freely available at https://www.danioffenlab.com/crispam.},
}
@article {pmid33014001,
year = {2020},
author = {Gramazio, P and Takayama, M and Ezura, H},
title = {Challenges and Prospects of New Plant Breeding Techniques for GABA Improvement in Crops: Tomato as an Example.},
journal = {Frontiers in plant science},
volume = {11},
number = {},
pages = {577980},
pmid = {33014001},
issn = {1664-462X},
abstract = {Over the last seven decades, γ-aminobutyric acid (GABA) has attracted great attention from scientists for its ubiquity in plants, animals and microorganisms and for its physiological implications as a signaling molecule involved in multiple pathways and processes. Recently, the food and pharmaceutical industries have also shown significantly increased interest in GABA, because of its great potential benefits for human health and the consumer demand for health-promoting functional compounds, resulting in the release of a plethora of GABA-enriched products. Nevertheless, many crop species accumulate appreciable GABA levels in their edible parts and could help to meet the daily recommended intake of GABA for promoting positive health effects. Therefore, plant breeders are devoting much effort into breeding elite varieties with improved GABA contents. In this regard, tomato (Solanum lycopersicum), the most produced and consumed vegetable worldwide and a fruit-bearing model crop, has received much consideration for its accumulation of remarkable GABA levels. Although many different strategies have been implemented, from classical crossbreeding to induced mutagenesis, new plant breeding techniques (NPBTs) have achieved the best GABA accumulation results in red ripe tomato fruits along with shedding light on GABA metabolism and gene functions. In this review, we summarize, analyze and compare all the studies that have substantially contributed to tomato GABA breeding with further discussion and proposals regarding the most recent NPBTs that could bring this process to the next level of precision and efficiency. This document also provides guidelines with which researchers of other crops might take advantage of the progress achieved in tomato for more efficient GABA breeding programs.},
}
@article {pmid33012992,
year = {2020},
author = {Damerum, A and Chapman, MA and Taylor, G},
title = {Innovative breeding technologies in lettuce for improved post-harvest quality.},
journal = {Postharvest biology and technology},
volume = {168},
number = {},
pages = {111266},
pmid = {33012992},
issn = {0925-5214},
abstract = {Societal awareness of healthy eating is increasing alongside the market for processed bagged salads, which remain as one of the strongest growing food sectors internationally, including most recently from indoor growing systems. Lettuce represents a significant proportion of this ready-to-eat salad market. However, such products typically have a short shelf life, with decay of post-harvest quality occurring through complex biochemical and physiological changes in leaves and resulting in spoilage, food waste and risks to health. We review the functional and quantitative genetic understanding of lettuce post-harvest quality, revealing that few findings have translated into improved cultivar development. We identify (i) phytonutrient status (for enhanced antioxidant and vitamin status, aroma and flavour) (ii) leaf biophysical, cell wall and water relations traits (for longer shelf life) (iii) leaf surface traits (for enhanced food safety and reduced spoilage) and (iv) chlorophyll, other pigments and developmental senescence traits (for appearance and colour), as key targets for future post-harvest breeding. Lettuce is well-placed for rapid future exploitation to address postharvest quality traits with extensive genomic resources including the recent release of the lettuce genome and the development of innovative breeding technologies. Although technologies such as CRISPR/Cas genome editing are paving the way for accelerated crop improvement, other equally important resources available for lettuce include extensive germplasm collections, bi-parental mapping and wide populations with genotyping for genomic selection strategies and extensive multiomic datasets for candidate gene discovery. We discuss current progress towards post-harvest quality breeding for lettuce and how such resources may be utilised for future crop improvement.},
}
@article {pmid33011993,
year = {2020},
author = {Gonçalves, BC and Lopes Barbosa, MG and Silva Olak, AP and Belebecha Terezo, N and Nishi, L and Watanabe, MA and Marinello, P and Zendrini Rechenchoski, D and Dejato Rocha, SP and Faccin-Galhardi, LC},
title = {Antiviral therapies: advances and perspectives.},
journal = {Fundamental &amp; clinical pharmacology},
volume = {},
number = {},
pages = {},
pmid = {33011993},
issn = {1472-8206},
abstract = {Viral infections cause high morbidity and mortality, threaten public health, and impose a socioeconomic burden. We have seen the recent emergence of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), the causative agent of COVID-19 that has already infected more than 29 million people, with more than 900 000 deaths since its identification in December 2019. Considering the significant impact of viral infections, research and development of new antivirals and control strategies are essential. In this paper, we summarize 96 antivirals approved by the Food and Drug Administration between 1987 and 2019. Of these, 49 (51%) are used in treatments against human immunodeficiency virus (HIV), four against human papillomavirus, six against cytomegalovirus, eight against hepatitis B virus, five against influenza, six against herpes simplex virus, 17 against hepatitis C virus and one against respiratory syncytial virus. This review also describes future perspectives for new antiviral therapies such as nanotechnologies, monoclonal antibodies and the CRISPR-Cas system. These strategies are suggested as inhibitors of viral replication by various means, such as direct binding to the viral particle, blocking the infection, changes in intracellular mechanisms or viral genes, preventing replication and virion formation. We also observed that a large number of viral agents have no therapy available and the majority of those approved in the last 32 years are restricted to some groups, especially anti-HIV. Additionally, the emergence of new viruses and strains resistant to available antivirals has necessitated the formulation of new antivirals.},
}
@article {pmid33011743,
year = {2020},
author = {Meaden, S and Capria, L and Alseth, E and Gandon, S and Biswas, A and Lenzi, L and van Houte, S and Westra, ER},
title = {Phage gene expression and host responses lead to infection-dependent costs of CRISPR immunity.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
pmid = {33011743},
issn = {1751-7370},
abstract = {CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.},
}
@article {pmid33011454,
year = {2020},
author = {Yang, Z and Blenner, M},
title = {Genome editing systems across yeast species.},
journal = {Current opinion in biotechnology},
volume = {66},
number = {},
pages = {255-266},
pmid = {33011454},
issn = {1879-0429},
support = {R35 GM133803/GM/NIGMS NIH HHS/United States ; },
abstract = {Yeasts are used to produce a myriad of value-added compounds. Engineering yeasts into cost-efficient cell factories is greatly facilitated by the availability of genome editing tools. While traditional engineering techniques such as homologous recombination-based gene knockout and pathway integration continue to be widely used, novel genome editing systems including multiplexed approaches, bacteriophage integrases, CRISPR-Cas systems, and base editors are emerging as more powerful toolsets to accomplish rapid genome scale engineering and phenotype screening. In this review, we summarized the techniques which have been successfully implemented in model yeast Saccharomyces cerevisiae as well as non-conventional yeast species. The mechanisms and applications of various genome engineering systems are discussed and general guidelines to expand genome editing systems from S. cerevisiae to other yeast species are also highlighted.},
}
@article {pmid33010669,
year = {2020},
author = {Seyed Hosseini, E and Riahi Kashani, N and Nikzad, H and Azadbakht, J and Hassani Bafrani, H and Haddad Kashani, H},
title = {The novel coronavirus Disease-2019 (COVID-19): Mechanism of action, detection and recent therapeutic strategies.},
journal = {Virology},
volume = {551},
number = {},
pages = {1-9},
doi = {10.1016/j.virol.2020.08.011},
pmid = {33010669},
issn = {1096-0341},
mesh = {Antiviral Agents/therapeutic use ; COVID-19/*diagnosis/*drug therapy/immunology/virology ; COVID-19 Testing ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; Humans ; Immunity ; Phylogeny ; SARS-CoV-2/*pathogenicity/*physiology/ultrastructure ; },
abstract = {Novel coronavirus SARS-CoV-2, designated as COVID-19 by the World Health Organization (WHO) on the February 11, 2020, is one of the highly pathogenic β-coronaviruses which infects human. Early diagnosis of COVID-19 is the most critical step to treat infection. The diagnostic tools are generally molecular methods, serology and viral culture. Recently CRISPR-based method has been investigated to diagnose and treat coronavirus infection. The emergence of 2019-nCoV during the influenza season, has led to the extensive use of antibiotics and neuraminidase enzyme inhibitors, taken orally and intravenously. Currently, antiviral inhibitors of SARS and MERS spike proteins, neuraminidase inhibitors, anti-inflammatory drugs and EK1 peptide are the available therapeutic options for SARS-CoV-2 infected individuals. In addition, Chloroquine, which was previously used for malarial and autoimmune disease, has shown efficacy in the 2019-nCoV infection treatment. In severe hypoxaemia, a combination of antibiotics, α-interferon, lopinavir and mechanical ventilation can effectively mitigate the symptoms. Comprehensive knowledge on the innate and adaptive immune responses, will make it possible to propose potent antiviral drugs with their effective therapeutic measures for the prevention of viral infection. This therapeutic strategy will help patients worldwide to protect themselves against severe and fatal viral infections, that potentially can evolve and develop drug resistance, and to reduce mortality rates.},
}
@article {pmid33009787,
year = {2020},
author = {Ushakova, VM and Morozova, AY and Reznik, AM and Kostyuk, GP and Chekhonin, VP},
title = {[Molecular Biological Aspects of Depressive Disorders: A Modern View].},
journal = {Molekuliarnaia biologiia},
volume = {54},
number = {5},
pages = {725-749},
doi = {10.31857/S0026898420050110},
pmid = {33009787},
issn = {0026-8984},
mesh = {Animals ; CRISPR-Cas Systems ; *Depressive Disorder/genetics ; Disease Models, Animal ; Gene Knockdown Techniques ; Humans ; Polymorphism, Genetic ; },
abstract = {Depression is a serious mental disorder that affects more than 300 million people worldwide. Due to the lack of effective treatment methods, the pathogenesis of depression is necessary to study in order to understand its development and find new therapies. The review describes the main mechanisms of depression, including the monoamine hypothesis, impairment of the hipotalamic-pituitary-adrenal axis, decreased production of neurotropic factors, and neuroinflammation. Genetic correlations, gene polymorphisms, and epigenetic mechanisms are also considered. Common and different features of the etiology are analyzed for depression and depressive conditions associated with other pathologies (schizophrenia, Parkinson disease, and Alzheimer's disease). Modern experimental methods used to investigate the molecular mechanisms of depressive conditions are described with a focus on gene knockouts in laboratory animals and the CRISPR/Cas technology. Consideration is given to optogenetic and chemogenetic methods and analyses of genetic polymorphisms and their combinations. The data may provide for a better integral understanding of the modern ideas about the pathogenesis of depression as an isolated or comorbid disorder and the prospects in studying the mechanisms of depressive conditions.},
}
@article {pmid33008045,
year = {2020},
author = {Hewes, AM and Sansbury, BM and Kmiec, EB},
title = {The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {33008045},
issn = {2073-4425},
support = {1588//BIRD Foundation/ ; P20GM103446//Delaware IDea Network of Biomedical Research Excellence and National Institutes of Health/National Institute of General Medical Sciences/ ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.},
}
@article {pmid33007045,
year = {2020},
author = {Thakur, VS and Welford, SM},
title = {Generation of a conditional mutant knock-in under the control of the natural promoter using CRISPR-Cas9 and Cre-Lox systems.},
journal = {PloS one},
volume = {15},
number = {10},
pages = {e0240256},
pmid = {33007045},
issn = {1932-6203},
support = {R01 CA187053/CA/NCI NIH HHS/United States ; },
mesh = {Blotting, Western ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; Genetic Vectors/genetics ; Humans ; Integrases/*genetics ; Promoter Regions, Genetic/genetics ; Recombination, Genetic/genetics ; },
abstract = {Modulation of gene activity by creating mutations has contributed significantly to the understanding of protein functions. Oftentimes, however, mutational analyses use overexpression studies, in which proteins are taken out of their normal contexts and stoichiometries. In the present work, we sought to develop an approach to simultaneously use the CRISPR/Cas9 and Cre-Lox techniques to modify the endogenous SAT1 gene to introduce mutant forms of the protein while still under the control of its natural gene promoter. We cloned the C-terminal portion of wild type (WT) SAT1, through the transcriptional stop elements, and flanked by LoxP sites in front of an identical version of SAT1 containing point mutations in critical binding sites. The construct was inserted into the endogenous SAT1 locus by Non-Homologous End Joining (NHEJ) after a CRISPR/Cas9 induced DNA double strand break. After validating that normal function of SAT1 was not altered by the insertional event, we were then able to assess the impact of point mutations by introduction of Cre recombinase. The system thus enables generation of cells in which endogenous WT SAT1 can be conditionally modified, and allow investigation of the functional consequences of site specific mutations in the context of the normal promoter and chromatin regulation.},
}
@article {pmid33006762,
year = {2020},
author = {Falk, MJ},
title = {The pursuit of precision mitochondrial medicine: Harnessing preclinical cellular and animal models to optimize mitochondrial disease therapeutic discovery.},
journal = {Journal of inherited metabolic disease},
volume = {},
number = {},
pages = {},
doi = {10.1002/jimd.12319},
pmid = {33006762},
issn = {1573-2665},
support = {R01-HD065858//National Institute of Child Health and Human Development (US)/ ; R01-GM120762//National Institute of General Medical Sciences (US)/ ; R35-GM134863//National Institute of General Medical Sciences (US)/ ; //Raptor Pharmaceuticals/ ; //The Children's Hospital of Philadelphia/ ; },
abstract = {Mitochondria share extensive evolutionary conservation across nearly all living species. This homology allows robust insights to be gained into pathophysiologic mechanisms and therapeutic targets for the heterogeneous class of primary mitochondrial diseases (PMDs) through the study of diverse in vitro cellular and in vivo animal models. Dramatic advances in genetic technologies, ranging from RNA interference to achieve graded knock-down of gene expression to CRISPR/Cas-based gene editing that yields a stable gene knock-out or targeted mutation knock-in, have enabled the ready establishment of mitochondrial disease models for a plethora of individual nuclear gene disorders. These models are complemented and extended by the use of pharmacologic inhibitor-based stressors to characterize variable degrees, onset, duration, and combinations of acute on chronic mitochondrial dysfunction in individual respiratory chain enzyme complexes or distinct biochemical pathways within mitochondria. Herein is described the rationale for, and progress made in, &quot;therapeutic cross-training,&quot; a novel approach meant to improve the validity and rigor of experimental conclusions when testing therapies by studying treatment effects in multiple, evolutionarily-distinct species, including Caenorhabditis elegans (invertebrate, worm), Danio rerio (vertebrate, zebrafish), Mus musculus (mammal, mouse), and/or human patient primary fibroblast cell line models of PMD. The goal of these preclinical studies is to identify lead therapies from candidate molecules or library screens that consistently demonstrate efficacy, with minimal toxicity, in specific subtypes of mitochondrial disease. Conservation of in vitro and in vivo therapeutic effects of lead molecules across species has proven extensive, where molar concentrations found to be toxic or efficacious in one species are often consistent with therapeutic effects at similar doses seen in other mitochondrial disease models. Phenotypic outcome studies in all models are prioritized at the level of survival and function, to reflect the ultimate goal of developing highly potent therapies for human mitochondrial disease. Lead compounds that demonstrate significant benefit on gross phenotypes may be further scrutinized in these same models to decipher their cellular targets, mechanism(s), and detailed biochemical effects. High-throughput, automated technologic advances will be discussed that enable efficient, parallel screening in a diverse array of mitochondrial disease disorders and overarching subclasses of compounds, concentrations, libraries, and combinations. Overall, this therapeutic cross-training approach has proven valuable to identify compounds with optimal potency and safety profiles among major biochemical subtypes or specific genetic etiologies of mitochondrial disease. This approach further supports rational prioritization of lead compounds, target concentrations, and specific disease phenotypes, outcomes, and subgroups to optimally inform the design of clinical trials that test their efficacy in human mitochondrial disease subjects.},
}
@article {pmid33005486,
year = {2020},
author = {Hoque, MN and Chaudhury, A and Akanda, MAM and Hossain, MA and Islam, MT},
title = {Genomic diversity and evolution, diagnosis, prevention, and therapeutics of the pandemic COVID-19 disease.},
journal = {PeerJ},
volume = {8},
number = {},
pages = {e9689},
pmid = {33005486},
issn = {2167-8359},
abstract = {The coronavirus disease 19 (COVID-19) is a highly transmittable and pathogenic viral infection caused by a novel evolutionarily divergent RNA virus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus first emerged in Wuhan, China in December 2019, and subsequently spreaded around the world. Genomic analyses revealed that this zoonotic virus may be evolved naturally but not a purposefully manipulated laboratory construct. However, currently available data are not sufficient to precisely conclude the origin of this fearsome virus. Comprehensive annotations of the whole-genomes revealed hundreds of nucleotides, and amino acids mutations, substitutions and/or deletions at different positions of the ever changing SARS-CoV-2 genome. The spike (S) glycoprotein of SARS-CoV-2 possesses a functional polybasic (furin) cleavage site at the S1-S2 boundary through the insertion of 12 nucleotides. It leads to the predicted acquisition of 3-O-linked glycan around the cleavage site. Although real-time RT-PCR methods targeting specific gene(s) have widely been used to diagnose the COVID-19 patients, however, recently developed more convenient, cheap, rapid, and specific diagnostic tools targeting antigens or CRISPR-Cas-mediated method or a newly developed plug and play method should be available for the resource-poor developing countries. A large number of candidate drugs, vaccines and therapies have shown great promise in early trials, however, these candidates of preventive or therapeutic agents have to pass a long path of trials before being released for the practical application against COVID-19. This review updates current knowledge on origin, genomic evolution, development of the diagnostic tools, and the preventive or therapeutic remedies of the COVID-19. We also discussed the future scopes for research, effective management, and surveillance of the newly emerged COVID-19 disease.},
}
@article {pmid33005303,
year = {2020},
author = {Xu, Y and Li, Z},
title = {CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy.},
journal = {Computational and structural biotechnology journal},
volume = {18},
number = {},
pages = {2401-2415},
pmid = {33005303},
issn = {2001-0370},
abstract = {Genome editing is the modification of genomic DNA at a specific target site in a wide variety of cell types and organisms, including insertion, deletion and replacement of DNA, resulting in inactivation of target genes, acquisition of novel genetic traits and correction of pathogenic gene mutations. Due to the advantages of simple design, low cost, high efficiency, good repeatability and short-cycle, CRISPR-Cas systems have become the most widely used genome editing technology in molecular biology laboratories all around the world. In this review, an overview of the CRISPR-Cas systems will be introduced, including the innovations, the applications in human disease research and gene therapy, as well as the challenges and opportunities that will be faced in the practical application of CRISPR-Cas systems.},
}
@article {pmid33004844,
year = {2020},
author = {de Melo, BP and Lourenço-Tessutti, IT and Paixão, JFR and Noriega, DD and Silva, MCM and de Almeida-Engler, J and Fontes, EPB and Grossi-de-Sa, MF},
title = {Transcriptional modulation of AREB-1 by CRISPRa improves plant physiological performance under severe water deficit.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {16231},
pmid = {33004844},
issn = {2045-2322},
mesh = {Arabidopsis/genetics/physiology ; Arabidopsis Proteins/genetics/*physiology ; Basic-Leucine Zipper Transcription Factors/genetics/*physiology ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Dehydration/genetics ; Gene Editing ; Gene Expression Regulation, Plant ; Plant Physiological Phenomena/*genetics ; Plants, Genetically Modified ; },
abstract = {Plants are sessile organisms, which are vulnerable to environmental stresses. As such, plants have developed multiple molecular, physiological, and cellular mechanisms to cope with natural stressors. However, these environmental adversities, including drought, are sources of the main agribusiness problems since they interfere with plant growth and productivity. Particularly under water deprivation conditions, the abscisic acid-responsive element-binding protein AREB1/ABF2 plays an important role in drought stress response and physiological adaptation. In this investigation, we provide substantial confirmation for the role of AREB1/ABF2 in plant survival under severe water deficit using the CRISPR activation (CRISPRa) technique to enhance the AREB1 gene expression. In our strategy, the inactive nuclease dCas9 was fused with an Arabidopsis histone acetyltransferase 1, which improves gene expression by remodeling chromatin. The AREB1 overexpression promotes an improvement in the physiological performance of the transgenic homozygous plants under drought, which was associated with an increase in chlorophyll content, antioxidant enzyme activity, and soluble sugar accumulation, leading to lower reactive oxygen species accumulation. Finally, we found that the CRISPR-mediated up-regulation of AREB1 changes the abundance of several downstream ABA-inducible genes, allowing us to report that CRISPRa dCas9-HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.},
}
@article {pmid33004521,
year = {2020},
author = {Miyawaki, S and Kuroki, S and Maeda, R and Okashita, N and Koopman, P and Tachibana, M},
title = {The mouse Sry locus harbors a cryptic exon that is essential for male sex determination.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6512},
pages = {121-124},
doi = {10.1126/science.abb6430},
pmid = {33004521},
issn = {1095-9203},
mesh = {Animals ; CRISPR-Cas Systems ; *Exons ; Gene Editing ; *Genes, Essential ; Genetic Loci ; Male ; Mice ; Mice, Transgenic ; RNA, Untranslated/genetics ; Sequence Deletion ; *Sex Determination Processes ; Sex-Determining Region Y Protein/*genetics ; Transcription, Genetic ; },
abstract = {The mammalian sex-determining gene Sry induces male development. Since its discovery 30 years ago, Sry has been believed to be a single-exon gene. Here, we identified a cryptic second exon of mouse Sry and a corresponding two-exon type Sry (Sry-T) transcript. XY mice lacking Sry-T were sex-reversed, and ectopic expression of Sry-T in XX mice induced male development. Sry-T messenger RNA is expressed similarly to that of canonical single-exon type Sry (Sry-S), but SRY-T protein is expressed predominantly because of the absence of a degron in the C terminus of SRY-S. Sry exon2 appears to have evolved recently in mice through acquisition of a retrotransposon-derived coding sequence to replace the degron. Our findings suggest that in nature, SRY-T, not SRY-S, is the bona fide testis-determining factor.},
}
@article {pmid33002715,
year = {2020},
author = {Ruiz-Hernández, UE and Pelcastre-Rodriguez, LI and Cabrero-Martínez, OA and Hernández-Cortez, C and Castro-Escarpulli, G},
title = {Analysis of CRISPR-Cas systems in Gardnerella suggests its potential role in the mechanisms of bacterial vaginosis.},
journal = {Computational biology and chemistry},
volume = {89},
number = {},
pages = {107381},
doi = {10.1016/j.compbiolchem.2020.107381},
pmid = {33002715},
issn = {1476-928X},
abstract = {Bacterial vaginosis (BV) is the principal cause of vaginal discharge among women, and it can lead to many comorbidities with a negative impact in women's daily activities. Despite the fact that the pathophysiological process of BV remains unclear, great advances had been achieved in determining consequences of the shift in the vaginal community, and it was defined that Gardnerella spp., plays a key role in the pathogenesis of BV. Interactions of vaginal phage communities and bacterial hosts may be relevant in eubiosis/dysbiosis states, so defense mechanisms in Gardnerella spp., against phage infections could be relevant in BV development. In this study, we analyzed CRISPR-Cas systems among the 13 Gardnerella species recently classified, considering that these systems act as prokaryotic immune systems against phages, plasmids, and other mobile genetic elements. In silico analyses for CRISPR-Cas systems mining over the 81 Gardnerella spp., strains genomes analyzed led to the identification of subtypes I-E and II-C. Spacers analyses showed a hypervariable region across species, providing a high resolution level in order to distinguish clonality in strains, which was supported with phylogenomic analyses based on Virtual Genomic Fingerprinting. Moreover, most of the spacers revealed interactions between Gardnerella spp., strains and prophages over the genus. Furthermore, virulence traits of the 13 species showed insights of potential niche specificity in the vaginal microbiome. Overall, our results suggest that the CRISPR-Cas systems in the genus Gardnerella may play an important role in the mechanisms of the development and maintenance of BV, considering that the Gardnerella species occupies different niches in the vaginal community; in addition, spacer sequences can be used for genotyping studies.},
}
@article {pmid33002419,
year = {2020},
author = {Neggers, JE and Jacquemyn, M and Dierckx, T and Kleinstiver, BP and Thibaut, HJ and Daelemans, D},
title = {enAsCas12a Enables CRISPR-Directed Evolution to Screen for Functional Drug Resistance Mutations in Sequences Inaccessible to SpCas9.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2020.09.025},
pmid = {33002419},
issn = {1525-0024},
abstract = {While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug's target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.},
}
@article {pmid32999368,
year = {2020},
author = {Leungtongkam, U and Thummeepak, R and Kitti, T and Tasanapak, K and Wongwigkarn, J and Styles, KM and Wellington, EMH and Millard, AD and Sagona, AP and Sitthisak, S},
title = {Genomic analysis reveals high virulence and antibiotic resistance amongst phage susceptible Acinetobacter baumannii.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {16154},
pmid = {32999368},
issn = {2045-2322},
mesh = {Acinetobacter Infections/*microbiology ; Acinetobacter baumannii/drug effects/*genetics ; Anti-Bacterial Agents/pharmacology ; Bacteriophages ; Biofilms/drug effects ; Drug Resistance, Bacterial/*genetics ; *Genome, Bacterial ; Virulence ; },
abstract = {In this study, we examined the association between antimicrobial resistance, CRISPR/Cas systems and virulence with phage susceptibility in Acinetobacter baumannii and investigated draft genomes of phage susceptible multidrug resistant A. baumannii strains from Thailand. We investigated 230 A. baumannii strains using 17 lytic A. baumannii phages and the phage susceptibility was 46.5% (107/230). Phage susceptibility was also associated with resistance to numerous antibiotics (p-value < 0.05). We also found association between biofilm formation and the presence of ompA gene among phage susceptible A. baumannii strains (p-value < 0.05). A. baumannii isolates carrying cas5 or combinations of two or three other cas genes, showed a significant increase in phage resistance. Whole-genome sequences of seven phage susceptible A. baumannii isolates revealed that six groups of antibiotic resistance genes were carried by all seven phage susceptible A. baumannii. All strains carried biofilm associated genes and two strains harbored complete prophages, acquired copper tolerance genes, and CRISPR-associated (cas) genes. In conclusion, our data exhibits an association between virulence determinants and biofilm formation among phage susceptible A. baumannii strains. These data help to understand the bacterial co-evolution with phages.},
}
@article {pmid32999292,
year = {2020},
author = {Nguyen, LT and Smith, BM and Jain, PK},
title = {Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4906},
doi = {10.1038/s41467-020-18615-1},
pmid = {32999292},
issn = {2041-1723},
mesh = {Bacterial Proteins/*metabolism ; Betacoronavirus/*genetics/isolation &amp; purification ; COVID-19 ; COVID-19 Testing ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems ; Clinical Laboratory Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/diagnosis/virology ; DNA, Single-Stranded ; Endodeoxyribonucleases/*metabolism ; Nucleic Acid Amplification Techniques/*methods ; Pandemics ; Pneumonia, Viral ; RNA, Guide/genetics ; RNA, Viral/genetics/*isolation &amp; purification ; SARS-CoV-2 ; Trans-Activators/*metabolism ; },
abstract = {The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3'- or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40-60 min.},
}
@article {pmid32997714,
year = {2020},
author = {Pöhler, M and Guttmann, S and Nadzemova, O and Lenders, M and Brand, E and Zibert, A and Schmidt, HH and Sandfort, V},
title = {CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B.},
journal = {PloS one},
volume = {15},
number = {9},
pages = {e0239411},
pmid = {32997714},
issn = {1932-6203},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Copper-Transporting ATPases/deficiency/*genetics ; Gene Editing/*methods ; Gene Knock-In Techniques ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; *Mutation ; },
abstract = {Wilson's disease (WD) is a monogenetic liver disease that is based on a mutation of the ATP7B gene and leads to a functional deterioration in copper (Cu) excretion in the liver. The excess Cu accumulates in various organs such as the liver and brain. WD patients show clinical heterogeneity, which can range from acute or chronic liver failure to neurological symptoms. The course of the disease can be improved by a life-long treatment with zinc or chelators such as D-penicillamine in a majority of patients, but serious side effects have been observed in a significant portion of patients, e.g. neurological deterioration and nephrotoxicity, so that a liver transplant would be inevitable. An alternative therapy option would be the genetic correction of the ATP7B gene. The novel gene therapy method CRISPR/Cas9, which has recently been used in the clinic, may represent a suitable therapeutic opportunity. In this study, we first initiated an artificial ATP7B point mutation in a human cell line using CRISPR/Cas9 gene editing, and corrected this mutation by the additional use of single-stranded oligo DNA nucleotides (ssODNs), simulating a gene correction of a WD point mutation in vitro. By the addition of 0.5 mM of Cu three days after lipofection, a high yield of CRISPR/Cas9-mediated ATP7B repaired cell clones was achieved (60%). Moreover, the repair efficiency was enhanced using ssODNs that incorporated three blocking mutations. The repaired cell clones showed a high resistance to Cu after exposure to increasing Cu concentrations. Our findings indicate that CRISPR/Cas9-mediated correction of ATP7B point mutations is feasible and may have the potential to be transferred to the clinic.},
}
@article {pmid32996421,
year = {2020},
author = {Watanabe, K and Nakamura, T and Onodera, S and Saito, A and Shibahara, T and Azuma, T},
title = {A novel GNAS-mutated human induced pluripotent stem cell model for understanding GNAS-mutated tumors.},
journal = {Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine},
volume = {42},
number = {9},
pages = {1010428320962588},
doi = {10.1177/1010428320962588},
pmid = {32996421},
issn = {1423-0380},
mesh = {Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Chromogranins/antagonists &amp; inhibitors/*genetics ; GTP-Binding Protein alpha Subunits, Gs/antagonists &amp; inhibitors/*genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism/*pathology ; Male ; Mice ; Mice, SCID ; *Models, Biological ; *Mutation ; Teratoma/genetics/*pathology ; },
abstract = {A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 (GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNASR201H-mutated (GNASR201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell-derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNASR201H-mutated induced pluripotent stem cells and GNASR201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNASR201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.},
}
@article {pmid32994412,
year = {2020},
author = {Lundin, A and Porritt, MJ and Jaiswal, H and Seeliger, F and Johansson, C and Bidar, AW and Badertscher, L and Wimberger, S and Davies, EJ and Hardaker, E and Martins, CP and James, E and Admyre, T and Taheri-Ghahfarokhi, A and Bradley, J and Schantz, A and Alaeimahabadi, B and Clausen, M and Xu, X and Mayr, LM and Nitsch, R and Bohlooly-Y, M and Barry, ST and Maresca, M},
title = {Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4903},
pmid = {32994412},
issn = {2041-1723},
mesh = {Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics ; Cell Line, Tumor ; Doxycycline/pharmacology ; Drug Discovery/*methods ; Drug Screening Assays, Antitumor/methods ; Female ; Gene Editing/*methods ; Gene Expression/drug effects/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Genetic Vectors/genetics ; HEK293 Cells ; High-Throughput Screening Assays/methods ; Humans ; Lung Neoplasms/*drug therapy/genetics ; Male ; Mice ; Mice, Transgenic ; RNA, Guide/genetics ; Recombination, Genetic/drug effects ; Reproducibility of Results ; Transcriptional Activation/drug effects ; Transfection/methods ; Transgenes/genetics ; },
abstract = {The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.},
}
@article {pmid32994292,
year = {2020},
author = {Solbiati, J and Duran-Pinedo, A and Godoy Rocha, F and Gibson, FC and Frias-Lopez, J},
title = {Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3.},
journal = {mSystems},
volume = {5},
number = {5},
pages = {},
pmid = {32994292},
issn = {2379-5077},
support = {R01 DE021553/DE/NIDCR NIH HHS/United States ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is a unique genomic entity that provides prokaryotic cells with adaptive and heritable immunity. Initial studies identified CRISPRs as central elements used by bacteria to protect against foreign nucleic acids; however, emerging evidence points to CRISPR involvement in bacterial virulence. The present study aimed to identify the participation of one CRISPR-Cas protein, Cas3, in the virulence of the oral pathogen Porphyromonas gingivalis, an organism highly associated with periodontitis. Our results show that compared to the wild type, a mutant with a deletion of the Cas3 gene, an essential nuclease part of the class 1 type I CRISPR-Cas system, increased the virulence of P. gingivalisIn vitro infection modeling revealed only mildly enhanced production of proinflammatory cytokines by THP-1 cells when infected with the mutant strain. Dual transcriptome sequencing (RNA-seq) analysis of infected THP-1 cells showed an increase in expression of genes associated with pathogenesis in response to Δcas3 mutant infection, with the target of Cas3 activities in neutrophil chemotaxis and gene silencing. The importance of cas3 in controlling virulence was corroborated in a Galleria mellonella infection model, where the presence of the Δcas3 mutant resulted in a statistically significant increase in mortality of G. mellonella A time-series analysis of transcription patterning during infection showed that G. mellonella elicited very different immune responses to the wild-type and the Δcas3 mutant strains and revealed a rearrangement of association in coexpression networks. Together, these observations show for the first time that Cas3 plays a significant role in regulating the virulence of P. gingivalisIMPORTANCEPorphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivaliscas3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection.},
}
@article {pmid32991234,
year = {2020},
author = {Zhang, Y and Marchisio, MA},
title = {Type II anti-CRISPR proteins as a new tool for synthetic biology.},
journal = {RNA biology},
volume = {},
number = {},
pages = {1-14},
doi = {10.1080/15476286.2020.1827803},
pmid = {32991234},
issn = {1555-8584},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable immune response against invading DNA. The discovery of anti-CRISPR proteins (Acrs), which are inhibitors of CRISPR-Cas, mainly encoded by phages and prophages, showed a co-evolution history between prokaryotes and phages. In the past decade, the CRISPR-Cas systems together with the corresponding Acrs have been turned into a genetic-engineering tool. Among the six types of CRISPR-Cas characterized so far, type II CRISPR-Cas system is the most popular in biotechnology. Here, we discuss about the discovery, the reported inhibitory mechanisms, and the applications in both gene editing and gene transcriptional regulation of type II Acrs. Moreover, we provide insights into future potential research and feasible applications.},
}
@article {pmid32990416,
year = {2020},
author = {Kim, GE and Lee, SY and Park, HH},
title = {A high-resolution (1.2 Å) crystal structure of the anti-CRISPR protein AcrIF9.},
journal = {FEBS open bio},
volume = {10},
number = {12},
pages = {2532-2540},
pmid = {32990416},
issn = {2211-5463},
support = {NRF-2018R1A4A1023822//National Research Foundation/ ; //Chung-Ang University/ ; },
abstract = {Prokaryotic adaptive immunity by CRISPR-Cas systems, which confer resistance to foreign genetic elements, has been used by bacteria to combat viruses. To cope, viruses evolved multiple anti-CRISPR proteins, which can inhibit system function through various mechanisms. Although the structures and mechanisms of several anti-CRISPR proteins have been elucidated, those of the AcrIF9 family have not yet been identified. To understand the molecular basis underlying AcrIF9 anti-CRISPR function, we determined the 1.2 Å crystal structure of AcrIF9. Structural and biochemical studies showed that AcrIF9 exists in monomeric form in solution and can directly interact with DNA using a positively charged cleft. Based on analysis of the structure, we suggest part of the anti-CRISPR molecular mechanism by AcrIF9.},
}
@article {pmid32989016,
year = {2020},
author = {Lin, Z and King, R and Tang, V and Myers, G and Balbin-Cuesta, G and Friedman, A and McGee, B and Desch, K and Ozel, AB and Siemieniak, D and Reddy, P and Emmer, B and Khoriaty, R},
title = {The Endoplasmic Reticulum Cargo Receptor SURF4 Facilitates Efficient Erythropoietin Secretion.},
journal = {Molecular and cellular biology},
volume = {40},
number = {23},
pages = {},
pmid = {32989016},
issn = {1098-5549},
support = {R01 HL141399/HL/NHLBI NIH HHS/United States ; R01 HL148333/HL/NHLBI NIH HHS/United States ; T32 CA009357/CA/NCI NIH HHS/United States ; T32 GM007315/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Endoplasmic Reticulum/*metabolism ; Erythropoiesis/*physiology ; Erythropoietin/analysis/*metabolism ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Membrane Proteins/genetics/*metabolism ; Protein Transport/physiology ; RNA, Guide/genetics ; },
abstract = {Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent single guide RNAs (sgRNAs) resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the endoplasmic reticulum (ER) compartment and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.},
}
@article {pmid32988588,
year = {2020},
author = {Lee, SY and Kim, GE and Kim, YG and Park, HH},
title = {A 1.3 Å high-resolution crystal structure of an anti-CRISPR protein, AcrI E2.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {4},
pages = {751-757},
doi = {10.1016/j.bbrc.2020.09.067},
pmid = {32988588},
issn = {1090-2104},
abstract = {As a result of bacterial infection with viruses, bacteria have developed CRISPR-Cas as an adaptive immune system, which allows them to destroy the viral genetic material introduced via infection. However, viruses have also evolved to develop multiple anti-CRISPR proteins, which are capable of inactivating the CRISPR-Cas adaptive immune system to combat bacteria. In this study, we aimed to elucidate the molecular mechanisms associated with anti-CRISPR proteins by determining a high-resolution crystal structure (1.3 Å) of Type I-E anti-CRISPR protein called AcrIE2. Our structural analysis revealed that AcrIE2 was composed of unique folds comprising five antiparallel β-sheets (β1∼β5) surrounding one α-helix (α1) in the order, β2β1α1β5β4β3. Structural comparison of AcrIE2 with a structural homolog called AcrIF9 showed that AcrIE2 contained a long and flexible β4-β5 connecting loop and a distinct surface feature. These results indicated that the inhibitory mechanism of AcrIE2 might be different from that of AcrIF9. This unique structure of AcrIE2 indicates its special mode of CRISPR-Cas inhibitory activity. Therefore, this study helps us understand the diversity in the inhibitory mechanisms of Acr family.},
}
@article {pmid32987327,
year = {2020},
author = {Li, CY and Zheng, B and Liu, YH and Gao, JL and Zheng, MQ and Pang, DW and Tang, HW},
title = {A boosting upconversion luminescent resonance energy transfer and biomimetic periodic chip integrated CRISPR/Cas12a biosensor for functional DNA regulated transduction of non-nucleic acid targets.},
journal = {Biosensors &amp; bioelectronics},
volume = {169},
number = {},
pages = {112650},
doi = {10.1016/j.bios.2020.112650},
pmid = {32987327},
issn = {1873-4235},
abstract = {Apart from gene editing capacity, the newly discovered CRISPR/Cas systems offer an exciting option for biosensing field because of their excellent target recognition accuracy. However, the currently constructed sensors are not only limited to nucleic acid analysis but also suffer from poor adaptability in complex samples and unsatisfying sensitivity. We herein introduce some advanced concepts to break through these bottlenecks. First, the sensing targets are extended by skillfully designing a functional DNA such as aptamer (for protein) and DNAzyme (for metal ion) to regulate the transduction of non-nucleic acid species and further activate the trans cleavage of CRISPR/Cas12a. Second, a boosting upconversion luminescent resonance energy is triggered by using a peculiar energy-confining notion, whereby the luminescence domain is intensively restricted in a very narrow space (~2.44 nm) and up to 92.9% of the green emission can be quenched by the approaching BHQ-1 modified reporters. Third, a bio-inspired periodic arrangement biomimetic chip (photonic crystal) is employed to selectively reflect the upconversion luminescence to achieve noteworthy signal enhancement (~35-fold). By utilizing very simple detection devices (a 980 nm portable laser and a smartphone), the CRISPR/Cas12a biosensor shows commendable sensitivity and specificity toward model targets (ATP and Na+, limits of detection are ~ 18 nM and ~0.37 μM, respectively). More importantly, the analysis of real complex samples demonstrate that the as-proposed platform can work as a powerful toolbox for monitoring the ATP fluctuation in single cell and point-of-care testing Na+ in human plasma, enabling a broad application prospect.},
}
@article {pmid32987180,
year = {2020},
author = {Lyu, C and Shi, H and Cui, Y and Li, M and Yan, Z and Yan, L and Jiang, Y},
title = {CRISPR-based biosensing is prospective for rapid and sensitive diagnosis of pediatric tuberculosis.},
journal = {International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases},
volume = {101},
number = {},
pages = {183-187},
doi = {10.1016/j.ijid.2020.09.1428},
pmid = {32987180},
issn = {1878-3511},
abstract = {Pediatric tuberculosis (TB) is an important part of global TB prevention and control. Diagnosis of childhood TB still remains challenging when using conventional tests, due to the non-specific clinical manifestations and paucibacillary nature of the specimens. Thus, a sensitive, rapid and low-cost diagnostic test is of great demand. Benefiting from specific and rapid Cas-protein-based catalytic activities, CRISPR-based biosensing platforms (CRISPR platforms) are showing superiority in detecting pathogen nucleic acid traces in clinical samples. Based on their excellent sensitivity, and time and cost saved in existing research, this study aimed to highlight the potential of CRISPR platforms as a tool for diagnosing pediatric TB, and advocate for studies to evaluate its performance in specimens collected from children, especially noninvasive specimens. These platforms are also promising in identifying drug resistance and genotyping. All of the above will help early diagnosis of pediatric TB, thus guide reasonable treatment, and be significant in achieving the World Health Organization End-TB strategy.},
}
@article {pmid32986839,
year = {2020},
author = {Ma, S and Wang, X and Hu, Y and Lv, J and Liu, C and Liao, K and Guo, X and Wang, D and Lin, Y and Rong, Z},
title = {Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {10590-10601},
pmid = {32986839},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Cholangiocarcinoma/*genetics/pathology ; DNA-Binding Proteins/genetics ; Gene Editing ; Gene Knock-In Techniques ; Genetic Therapy ; Humans ; Mice ; Oncogene Proteins, Fusion/genetics ; Protein Domains/genetics ; Proto-Oncogene Proteins p21(ras)/*genetics ; RNA, Guide ; RNA, Untranslated/genetics ; Receptors, Antigen, T-Cell/*genetics ; T-Lymphocytes/metabolism/pathology ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.},
}
@article {pmid32986708,
year = {2020},
author = {Gao, Y and Liu, YC and Jia, SZ and Liang, YT and Tang, Y and Xu, YS and Kawasaki, H and Wang, HB},
title = {Imaginal disc growth factor maintains cuticle structure and controls melanization in the spot pattern formation of Bombyx mori.},
journal = {PLoS genetics},
volume = {16},
number = {9},
pages = {e1008980},
pmid = {32986708},
issn = {1553-7404},
mesh = {Animals ; Bombyx/anatomy &amp; histology/*physiology ; CRISPR-Cas Systems ; Gene Expression Regulation ; Gene Knockout Techniques ; Insect Proteins/genetics/*metabolism ; Larva/genetics/physiology ; Melanins/biosynthesis/genetics/*metabolism ; Metabolomics/methods ; Mutation ; Phylogeny ; Pigmentation/*physiology ; Transcription Factors/genetics/metabolism ; },
abstract = {The complex stripes and patterns of insects play key roles in behavior and ecology. However, the fine-scale regulation mechanisms underlying pigment formation and morphological divergence remain largely unelucidated. Here we demonstrated that imaginal disc growth factor (IDGF) maintains cuticle structure and controls melanization in spot pattern formation of Bombyx mori. Moreover, our knockout experiments showed that IDGF is suggested to impact the expression levels of the ecdysone inducible transcription factor E75A and pleiotropic factors apt-like and Toll8/spz3, to further control the melanin metabolism. Furthermore, the untargeted metabolomics analyses revealed that BmIDGF significantly affected critical metabolites involved in phenylalanine, beta-alanine, purine, and tyrosine metabolism pathways. Our findings highlighted not only the universal function of IDGF to the maintenance of normal cuticle structure but also an underexplored space in the gene function affecting melanin formation. Therefore, this study furthers our understanding of insect pigment metabolism and melanin pattern polymorphisms.},
}
@article {pmid32986696,
year = {2020},
author = {Gai, T and Tong, X and Han, M and Li, C and Fang, C and Zou, Y and Hu, H and Xiang, H and Xiang, Z and Lu, C and Dai, F},
title = {Cocoonase is indispensable for Lepidoptera insects breaking the sealed cocoon.},
journal = {PLoS genetics},
volume = {16},
number = {9},
pages = {e1009004},
pmid = {32986696},
issn = {1553-7404},
mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*enzymology/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Homozygote ; Life Cycle Stages/*physiology ; Mutation ; Phylogeny ; Selection, Genetic ; Species Specificity ; },
abstract = {Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons.},
}
@article {pmid32986041,
year = {2020},
author = {Meuti, ME and Harrell, R},
title = {Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {163},
pages = {},
doi = {10.3791/61651},
pmid = {32986041},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Culex/*embryology/genetics ; Female ; Gene Editing ; Loss of Function Mutation ; Mosquito Vectors ; *Mutation ; West Nile virus ; },
abstract = {Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.},
}
@article {pmid32983033,
year = {2020},
author = {Ye, Q and Zhao, X and Liu, J and Zeng, Z and Zhang, Z and Liu, T and Li, Y and Han, W and Peng, N},
title = {CRISPR-Associated Factor Csa3b Regulates CRISPR Adaptation and Cmr-Mediated RNA Interference in Sulfolobus islandicus.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {2038},
pmid = {32983033},
issn = {1664-302X},
abstract = {Acquisition of spacers confers the CRISPR-Cas system with the memory to defend against invading mobile genetic elements. We previously reported that the CRISPR-associated factor Csa3a triggers CRISPR adaptation in Sulfolobus islandicus. However, a feedback regulation of CRISPR adaptation remains unclear. Here we show that another CRISPR-associated factor, Csa3b, binds a cyclic oligoadenylate (cOA) analog (5'-CAAAA-3') and mutation at its CARF domain, which reduces the binding affinity. Csa3b also binds the promoter of adaptation cas genes, and the cOA analog enhances their binding probably by allosteric regulation. Deletion of the csa3b gene triggers spacer acquisition from both plasmid and viral DNAs, indicating that Csa3b acted as a repressor for CRISPR adaptation. Moreover, we also find that Csa3b activates the expression of subtype cmr-α and cmr-β genes according to transcriptome data and demonstrate that Csa3b binds the promoters of cmr genes. The deletion of the csa3b gene reduces Cmr-mediated RNA interference activity, indicating that Csa3b acts as a transcriptional activator for Cmr-mediated RNA interference. In summary, our findings reveal a novel pathway for the regulation of CRISPR adaptation and CRISPR-Cmr RNA interference in S. islandicus. Our results also suggest a feedback repression of CRIPSR adaptation by the Csa3b factor and the cOA signal produced by the Cmr complex at the CRISPR interference stage.},
}
@article {pmid32980951,
year = {2020},
author = {Ma, D and Endo, S and Betsuyaku, S and Shimotohno, A and Fukuda, H},
title = {CLE2 regulates light-dependent carbohydrate metabolism in Arabidopsis shoots.},
journal = {Plant molecular biology},
volume = {104},
number = {6},
pages = {561-574},
doi = {10.1007/s11103-020-01059-y},
pmid = {32980951},
issn = {1573-5028},
support = {15H05958//Ministry of Education, Culture, Sports, Science and Technology/ ; 16H06377//Japan Society for the Promotion of Science/ ; },
mesh = {Arabidopsis/genetics/*metabolism/radiation effects ; Arabidopsis Proteins/genetics/*physiology ; CRISPR-Cas Systems ; *Carbohydrate Metabolism ; Darkness ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Gene-Environment Interaction ; Light ; Plant Shoots/*metabolism/radiation effects ; Plants, Genetically Modified ; Protein Domains/genetics ; Signal Transduction ; },
abstract = {KEY MESSAGE: This study focused on the role of CLE1-CLE7 peptides as environmental mediators and indicated that root-induced CLE2 functions systemically in light-dependent carbohydrate metabolism in shoots. Plants sense environmental stimuli and convert them into cellular signals, which are transmitted to distinct cells and tissues to induce adequate responses. Plant hormones and small secretory peptides often function as environmental stress mediators. In this study, we investigated whether CLAVATA3/EMBRYO SURROUNDING REGION-RELATED proteins, CLE1-CLE7, which share closely related CLE domains, mediate environmental stimuli in Arabidopsis thaliana. Expression analysis of CLE1-CLE7 revealed that these genes respond to different environmental stimuli, such as nitrogen deprivation, nitrogen replenishment, cold, salt, dark, and sugar starvation, in a sophisticated manner. To further investigate the function of CLE2, we generated transgenic Arabidopsis lines expressing the β-glucuronidase gene under the control of the CLE2 promoter or expressing the CLE2 gene under the control of an estradiol-inducible promoter. We also generated cle2-1 and cle2-2 mutants using the CRISPR/Cas9 technology. In these transgenic lines, dark induced the expression of CLE2 in the root vasculature. Additionally, induction of CLE2 in roots induced the expression of various genes not only in roots but also in shoots, and genes related to light-dependent carbohydrate metabolism were particularly induced in shoots. In addition, cle2 mutant plants showed chlorosis when subjected to a shade treatment. These results suggest that root-induced CLE2 functions systemically in light-dependent carbohydrate metabolism in shoots.},
}
@article {pmid32980369,
year = {2020},
author = {Bhat, MA and Bhat, MA and Kumar, V and Wani, IA and Bashir, H and Shah, AA and Rahman, S and Jan, AT},
title = {The era of editing plant genomes using CRISPR/Cas: A critical appraisal.},
journal = {Journal of biotechnology},
volume = {324},
number = {},
pages = {34-60},
doi = {10.1016/j.jbiotec.2020.09.013},
pmid = {32980369},
issn = {1873-4863},
abstract = {The versatility of Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) genome editing tool ushered biologists into an exciting era of editing genomes with great efficiency and at a pace that was never imagined before. Though the CRISPR/Cas genome editing was developed after Zinc Finger Nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs), it is more popular and successful than these genome editing systems. The advent of targetable nucleases such as Cas9 has enabled manipulation of genomes in an accurate and precise manner. The CRISPR/Cas system of editing plant genomes has technical and economical advantages over conventional breeding methods. It has led to the development of traits within plant genomes that fulfill the needs of mankind. Advent of innovative procedures have paved the way for effective and efficient genome editing that has revolutionized genetic aspects and meets the safety regulations toward development of crops. The present review highlights the critical aspects of employing CRISPR/Cas for editing plant genomes in comparison with previously known editing approaches, such as ZFNs and TALENs. The study includes descriptive information on the approaches, procedural programs and applications in editing plant genomes for improving traits such as crop yield, resistance against emerging pathogens, abiotic stresses and herbicide tolerance thereof in the present-day world.},
}
@article {pmid32979593,
year = {2020},
author = {Chen, Y and Shi, Y and Chen, Y and Yang, Z and Wu, H and Zhou, Z and Li, J and Ping, J and He, L and Shen, H and Chen, Z and Wu, J and Yu, Y and Zhang, Y and Chen, H},
title = {Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {169},
number = {},
pages = {112642},
doi = {10.1016/j.bios.2020.112642},
pmid = {32979593},
issn = {1873-4235},
mesh = {Betacoronavirus/genetics/*isolation &amp; purification ; Biosensing Techniques/instrumentation/*methods ; COVID-19 ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/*diagnosis/virology ; Equipment Design ; Fluorescence ; Humans ; Molecular Diagnostic Techniques/instrumentation/*methods ; Nucleic Acid Amplification Techniques/instrumentation/*methods ; Pandemics ; Pneumonia, Viral/*diagnosis/virology ; *Point-of-Care Systems ; SARS-CoV-2 ; Sensitivity and Specificity ; Smartphone ; },
abstract = {The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.},
}
@article {pmid32978771,
year = {2020},
author = {Jamehdor, S and Zaboli, KA and Naserian, S and Thekkiniath, J and Omidy, HA and Teimoori, A and Johari, B and Taromchi, AH and Sasano, Y and Kaboli, S},
title = {An overview of applications of CRISPR-Cas technologies in biomedical engineering.},
journal = {Folia histochemica et cytobiologica},
volume = {58},
number = {3},
pages = {163-173},
doi = {10.5603/FHC.a2020.0023},
pmid = {32978771},
issn = {1897-5631},
abstract = {Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one of the major genome editing systems and allows changing DNA levels of an organism. Among several CRISPR categories, the CRISPR-Cas9 system has shown a remarkable progression rate over its lifetime. Recently, other tools including CRISPR-Cas12 and CRISPR-Cas13 have been introduced. CRISPR-Cas9 system has played a key role in the industrial cell factory's production and improved our understanding of genome function. Additionally, this system has been used as one of the major genome editing systems for the diagnosis and treatment of several infectious and non-infectious diseases. In this review, we discuss CRISPR biology, its versatility, and its application in biomedical engineering.},
}
@article {pmid32978399,
year = {2020},
author = {Nguyen Tran, MT and Mohd Khalid, MKN and Wang, Q and Walker, JKR and Lidgerwood, GE and Dilworth, KL and Lisowski, L and Pébay, A and Hewitt, AW},
title = {Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4871},
pmid = {32978399},
issn = {2041-1723},
mesh = {Adenine/*chemistry ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cytosine ; DNA ; Exome ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome ; HEK293 Cells ; Humans ; RNA/*metabolism ; RNA Editing ; },
abstract = {Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.},
}
@article {pmid32977396,
year = {2020},
author = {Ates, I and Rathbone, T and Stuart, C and Bridges, PH and Cottle, RN},
title = {Delivery Approaches for Therapeutic Genome Editing and Challenges.},
journal = {Genes},
volume = {11},
number = {10},
pages = {},
pmid = {32977396},
issn = {2073-4425},
support = {P20 GM103499/GM/NIGMS NIH HHS/United States ; 1422921//SC TRIMH/ ; 5P20GM103499-16//SC INBRE/ ; },
abstract = {Impressive therapeutic advances have been possible through the advent of zinc-finger nucleases and transcription activator-like effector nucleases. However, discovery of the more efficient and highly tailorable clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas9) has provided unprecedented gene-editing capabilities for treatment of various inherited and acquired diseases. Despite recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In this review, we elaborate on the challenges and provide practical considerations for improving gene editing. Specifically, we highlight issues associated with delivery of gene-editing tools into clinically relevant cells.},
}
@article {pmid32976930,
year = {2020},
author = {Arya, SS and Rookes, JE and Cahill, DM and Lenka, SK},
title = {Next-generation metabolic engineering approaches towards development of plant cell suspension cultures as specialized metabolite producing biofactories.},
journal = {Biotechnology advances},
volume = {45},
number = {},
pages = {107635},
doi = {10.1016/j.biotechadv.2020.107635},
pmid = {32976930},
issn = {1873-1899},
abstract = {Plant cell suspension culture (PCSC) has emerged as a viable technology to produce plant specialized metabolites (PSM). While Taxol® and ginsenoside are two examples of successfully commercialized PCSC-derived PSM, widespread utilization of the PCSC platform has yet to be realized primarily due to a lack of understanding of the molecular genetics of PSM biosynthesis. Recent advances in computational, molecular and synthetic biology tools provide the opportunity to rapidly characterize and harness the specialized metabolic potential of plants. Here, we discuss the prospects of integrating computational modeling, artificial intelligence, and precision genome editing (CRISPR/Cas and its variants) toolboxes to discover the genetic regulators of PSM. We also explore how synthetic biology can be applied to develop metabolically optimized PSM-producing native and heterologous PCSC systems. Taken together, this review provides an interdisciplinary approach to realize and link the potential of next-generation computational and molecular tools to convert PCSC into commercially viable PSM-producing biofactories.},
}
@article {pmid32975299,
year = {2020},
author = {Poljak, M},
title = {Simplification of hepatitis C testing: a time to act.},
journal = {Acta dermatovenerologica Alpina, Pannonica, et Adriatica},
volume = {29},
number = {3},
pages = {129-132},
pmid = {32975299},
issn = {1581-2979},
abstract = {Hepatitis C virus (HCV) affects 71 million people worldwide. The development of reliable diagnostic tools in the last 2 decades and recent implementation of highly potent and safe antiviral drug combinations have paved the way to potential elimination of HCV as a public health threat by 2030. This article briefly discusses current and upcoming solutions for simplification of HCV testing taking into account the most recent guidance documents issued by major professional societies. The general consensus is that all patients with suspected HCV infection should be tested for anti-HCV antibodies as a first-line diagnostic test. Some anti-HCV rapid diagnostic tests have already attained the performance of standard anti-HCV enzyme immunoassays. If anti-HCV antibodies are detected, the presence of HCV RNA or HCV core antigen should be determined to identify patients with ongoing infection. Several innovative devices for detecting HCV in serum or plasma are in the late stages of development and are based on loop-mediated isothermal amplification, smartphone-operated instruments, biosensors, lab-on-a-chip solutions, paper-based microfluidics, and CRISPR-Cas. An important solution for low- and middle-income countries is the availability of HCV tests that could perform equally reliably from whole blood and dried blood spots as well as from serum or plasma. Another interesting diagnostic concept for these countries is near-to-patient diagnostics using mobile microbiological laboratories, following either the lab-on-a-drone or rent-a-point-of-care-test concepts. Using current and upcoming diagnostic approaches, the elimination of HCV is plausible, but in several countries this is probably not possible within the timeframe suggested by the World Health Organization. Two different elimination approaches have already been successfully tested in real-life conditions: micro-elimination and macro-elimination. The micro-elimination approach has resulted in successful elimination in specific population subgroups in some high-income countries. In at least two countries, Georgia and Egypt, a macro-elimination approach has shown impressive real-life results relatively quickly.},
}
@article {pmid32974560,
year = {2019},
author = {Yan, Y and Finnigan, GC},
title = {Analysis of CRISPR gene drive design in budding yeast.},
journal = {Access microbiology},
volume = {1},
number = {9},
pages = {e000059},
pmid = {32974560},
issn = {2516-8290},
abstract = {Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology - a gene drive - has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in yeast and fly model systems have developed a number of molecular safeguards to increase biosafety and control over drive systems in vivo, including titration of nuclease activity, anti-CRISPR-dependent inhibition and use of non-native DNA target sites. We have developed a CRISPR/Cas9 gene drive in Saccharomyces cerevisiae that allows for the safe and rapid examination of alternative drive designs and control mechanisms. In this study, we tested whether non-homologous end-joining (NHEJ) had occurred within diploid cells displaying a loss of the target allele following drive activation and did not detect any instances of NHEJ within multiple sampled populations. We also demonstrated successful multiplexing using two additional non-native target sequences. Furthermore, we extended our analysis of 'resistant' clones that still harboured both the drive and target selection markers following expression of Streptococcus pyogenes Cas9; de novo mutation or NHEJ-based repair could not explain the majority of these heterozygous clones. Finally, we developed a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus with a near 100 % success rate; resistant clones in this system could also be reactivated during a second round of Cas9 induction.},
}
@article {pmid32974171,
year = {2020},
author = {Chasov, V and Mirgayazova, R and Zmievskaya, E and Khadiullina, R and Valiullina, A and Stephenson Clarke, J and Rizvanov, A and Baud, MGJ and Bulatov, E},
title = {Key Players in the Mutant p53 Team: Small Molecules, Gene Editing, Immunotherapy.},
journal = {Frontiers in oncology},
volume = {10},
number = {},
pages = {1460},
pmid = {32974171},
issn = {2234-943X},
abstract = {The transcription factor p53 is a key tumor suppressor that is inactivated in almost all cancers due to either point mutations in the TP53 gene or overexpression of its negative regulators. The p53 protein is known as the &quot;cellular gatekeeper&quot; for its roles in facilitating DNA repair, cell cycle arrest or apoptosis upon DNA damage. Most p53 mutations are missense and result in either structural destabilization of the protein, causing its partial unfolding and deactivation under physiological conditions, or impairment of its DNA-binding properties. Tumor cells with p53 mutations are generally more immunogenic due to &quot;hot spot&quot; neoantigens that instigate the immune system response. In this review, we discuss the key therapeutic strategies targeting mutant p53 tumors, including classical approaches based on small molecule intervention and emerging technologies such as gene editing and T cell immunotherapy.},
}
@article {pmid32973430,
year = {2020},
author = {Gallego, C and Gonçalves, MAFV and Wijnholds, J},
title = {Novel Therapeutic Approaches for the Treatment of Retinal Degenerative Diseases: Focus on CRISPR/Cas-Based Gene Editing.},
journal = {Frontiers in neuroscience},
volume = {14},
number = {},
pages = {838},
pmid = {32973430},
issn = {1662-4548},
abstract = {Inherited retinal diseases encompass a highly heterogenous group of disorders caused by a wide range of genetic variants and with diverse clinical symptoms that converge in the common trait of retinal degeneration. Indeed, mutations in over 270 genes have been associated with some form of retinal degenerative phenotype. Given the immune privileged status of the eye, cell replacement and gene augmentation therapies have been envisioned. While some of these approaches, such as delivery of genes through recombinant adeno-associated viral vectors, have been successfully tested in clinical trials, not all patients will benefit from current advancements due to their underlying genotype or phenotypic traits. Gene editing arises as an alternative therapeutic strategy seeking to correct mutations at the endogenous locus and rescue normal gene expression. Hence, gene editing technologies can in principle be tailored for treating retinal degeneration. Here we provide an overview of the different gene editing strategies that are being developed to overcome the challenges imposed by the post-mitotic nature of retinal cell types. We further discuss their advantages and drawbacks as well as the hurdles for their implementation in treating retinal diseases, which include the broad range of mutations and, in some instances, the size of the affected genes. Although therapeutic gene editing is at an early stage of development, it has the potential of enriching the portfolio of personalized molecular medicines directed at treating genetic diseases.},
}
@article {pmid32973401,
year = {2020},
author = {Miri, SM and Tafsiri, E and Cho, WCS and Ghaemi, A},
title = {CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy.},
journal = {Cancer cell international},
volume = {20},
number = {},
pages = {456},
pmid = {32973401},
issn = {1475-2867},
abstract = {Cancer immunotherapy has been emerged as a promising strategy for treatment of a broad spectrum of malignancies ranging from hematological to solid tumors. One of the principal approaches of cancer immunotherapy is transfer of natural or engineered tumor-specific T-cells into patients, a so called &quot;adoptive cell transfer&quot;, or ACT, process. Construction of allogeneic T-cells is dependent on the employment of a gene-editing tool to modify donor-extracted T-cells and prepare them to specifically act against tumor cells with enhanced function and durability and least side-effects. In this context, CRISPR technology can be used to produce universal T-cells, equipped with recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR), through multiplex genome engineering using Cas nucleases. The robust potential of CRISPR-Cas in preparing the building blocks of ACT immunotherapy has broaden the application of such therapies and some of them have gotten FDA approvals. Here, we have collected the last investigations in the field of immuno-oncology conducted in partnership with CRISPR technology. In addition, studies that have addressed the challenges in the path of CRISPR-mediated cancer immunotherapy, as well as pre-treatment applications of CRISPR-Cas have been mentioned in detail.},
}
@article {pmid32973356,
year = {2020},
author = {Zhu, H and Li, C and Gao, C},
title = {Applications of CRISPR-Cas in agriculture and plant biotechnology.},
journal = {Nature reviews. Molecular cell biology},
volume = {21},
number = {11},
pages = {661-677},
doi = {10.1038/s41580-020-00288-9},
pmid = {32973356},
issn = {1471-0080},
abstract = {The prokaryote-derived CRISPR-Cas genome editing technology has altered plant molecular biology beyond all expectations. Characterized by robustness and high target specificity and programmability, CRISPR-Cas allows precise genetic manipulation of crop species, which provides the opportunity to create germplasms with beneficial traits and to develop novel, more sustainable agricultural systems. Furthermore, the numerous emerging biotechnologies based on CRISPR-Cas platforms have expanded the toolbox of fundamental research and plant synthetic biology. In this Review, we first briefly describe gene editing by CRISPR-Cas, focusing on the newest, precise gene editing technologies such as base editing and prime editing. We then discuss the most important applications of CRISPR-Cas in increasing plant yield, quality, disease resistance and herbicide resistance, breeding and accelerated domestication. We also highlight the most recent breakthroughs in CRISPR-Cas-related plant biotechnologies, including CRISPR-Cas reagent delivery, gene regulation, multiplexed gene editing and mutagenesis and directed evolution technologies. Finally, we discuss prospective applications of this game-changing technology.},
}
@article {pmid32973188,
year = {2020},
author = {Moro, LN and Viale, DL and Bastón, JI and Arnold, V and Suvá, M and Wiedenmann, E and Olguín, M and Miriuka, S and Vichera, G},
title = {Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {15587},
pmid = {32973188},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified/*genetics ; Base Sequence ; *CRISPR-Cas Systems ; Embryo, Mammalian/cytology/*metabolism ; Female ; Fibroblasts/cytology/metabolism ; *Gene Editing ; Gene Knockout Techniques/*veterinary ; Horses ; Mutation ; Myostatin/antagonists &amp; inhibitors/*genetics ; Nuclear Transfer Techniques/*veterinary ; Sequence Homology ; },
abstract = {The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.},
}
@article {pmid32973160,
year = {2020},
author = {He, X and Chen, Y and Beltran, DG and Kelly, M and Ma, B and Lawrie, J and Wang, F and Dodds, E and Zhang, L and Guo, J and Niu, W},
title = {Functional genetic encoding of sulfotyrosine in mammalian cells.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4820},
pmid = {32973160},
issn = {2041-1723},
support = {P30 GM103335/GM/NIGMS NIH HHS/United States ; R01 AI111862/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chemokines/metabolism ; Crystallography, X-Ray ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Ligands ; Models, Molecular ; Protein Conformation ; Receptors, CXCR4/genetics/metabolism ; Tyrosine/*analogs &amp; derivatives/*genetics/*metabolism ; Tyrosine-tRNA Ligase/chemistry/*genetics/*metabolism ; },
abstract = {Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.},
}
@article {pmid32972752,
year = {2020},
author = {Na, HH and Moon, S and Kim, KC},
title = {Knockout of SETDB1 gene using the CRISPR/cas-9 system increases migration and transforming activities via complex regulations of E-cadherin, β-catenin, STAT3, and Akt.},
journal = {Biochemical and biophysical research communications},
volume = {533},
number = {3},
pages = {486-492},
doi = {10.1016/j.bbrc.2020.09.026},
pmid = {32972752},
issn = {1090-2104},
abstract = {SETDB1 HMTase participates in various cellular processes via epigenetic transcriptional regulation. SETDB1 expression is downregulated by anticancer drug treatment in cancer cells, but we still need to verify the functional significance on SETDB1 downregulation. CRISPR/cas9 is a useful technology for doing a knockout (KO) of a target gene. It is widely used to examine the function of genes. In this study, we prepared SETDB1-KO from A549 human lung cancer cells using the CRISPR/Cas9 system, and we compared molecular changes between the A549 cells and the SETDB1-KO cells. The SETDB1-KO cell proliferation rate was slightly decreased as compared to the A549 cells, but there was no large difference in sensitivity with doxorubicin treatment. Instead, the migration activity and transforming activity were dramatically increased in SETDB-KO cells. Using a western blot analysis and an immunostaining experiment, we confirmed that SETDB1-KO downregulates the expression of E-cadherin and β-catenin. A qPCR and an RT-PCR analysis suggested that SETDB1 transcriptionally regulates E-cadherin and β-catenin. Moreover, E-cadherin expression was also detected in the cytoplasmic region of SETDB1-KO cells, indicating that functional localization of E-cadherin might be changed in SETDB1-KO cells. On the other hand, total levels of STAT3 and Akt were increased in the SETDB1-KO cells, but activation of STAT3 (pSTAT3) was not induced in doxorubicin-treated SETDB1-KO cells. SETDB1 overexpression into SETDB1-KO cells restores the expression of E-cadherin, β-catenin, STAT3, and Akt, suggesting that those proteins are tightly regulated by SETDB1. Collectively, we suggest that complex regulations on E-cadherin, β-catenin, STAT3, and Akt are correlated with the increased migration and transforming activity of SETDB1-KO cells.},
}
@article {pmid32969481,
year = {2020},
author = {Järås, M},
title = {.},
journal = {Lakartidningen},
volume = {117},
number = {},
pages = {},
pmid = {32969481},
issn = {1652-7518},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; },
}
@article {pmid32968780,
year = {2020},
author = {Değirmenci, L and Geiger, D and Rogé Ferreira, FL and Keller, A and Krischke, B and Beye, M and Steffan-Dewenter, I and Scheiner, R},
title = {CRISPR/Cas 9-Mediated Mutations as a New Tool for Studying Taste in Honeybees.},
journal = {Chemical senses},
volume = {45},
number = {8},
pages = {655-666},
doi = {10.1093/chemse/bjaa063},
pmid = {32968780},
issn = {1464-3553},
abstract = {Honeybees rely on nectar as their main source of carbohydrates. Sucrose, glucose, and fructose are the main components of plant nectars. Intriguingly, honeybees express only 3 putative sugar receptors (AmGr1, AmGr2, and AmGr3), which is in stark contrast to many other insects and vertebrates. The sugar receptors are only partially characterized. AmGr1 detects different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while AmGr3 is assumedly a fructose receptor. We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioral change and serve to characterize the function of taste receptors in vivo. CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in vivo. Biophysical receptor characterization in Xenopus oocytes and nonsense mutation of AmGr3 in honeybees unequivocally demonstrate that this receptor is highly specific for fructose.},
}
@article {pmid32968154,
year = {2020},
author = {Seruggia, D and Fernández, A and Cantero, M and Fernández-Miñán, A and Gomez-Skarmeta, JL and Pelczar, P and Montoliu, L},
title = {Boundary sequences flanking the mouse tyrosinase locus ensure faithful pattern of gene expression.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {15494},
pmid = {32968154},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Chromatin/metabolism/ultrastructure ; Enhancer Elements, Genetic/genetics ; Gene Editing ; Gene Expression Regulation/*genetics ; Genetic Loci/*genetics ; HEK293 Cells ; Humans ; Mice ; Monophenol Monooxygenase/*genetics/metabolism ; Promoter Regions, Genetic/genetics ; Zebrafish/embryology/genetics ; },
abstract = {Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators. While promoters and enhancers convey cell-type specific activating signals, insulators prevent the cross-talk of regulatory elements within adjacent loci and safeguard the specificity of action of promoters and enhancers towards their targets in a tissue specific manner. Using the mouse tyrosinase (Tyr) locus as an experimental model, a gene whose mutations are associated with albinism, we described the chromatin structure in cells at two distinct transcriptional states. Guided by chromatin structure, through the use of Chromosome Conformation Capture (3C), we identified sequences at the 5' and 3' boundaries of this mammalian gene that function as enhancers and insulators. By CRISPR/Cas9-mediated chromosomal deletion, we dissected the functions of these two regulatory elements in vivo in the mouse, at the endogenous chromosomal context, and proved their mechanistic role as genomic insulators, shielding the Tyr locus from the expression patterns of adjacent genes.},
}
@article {pmid32968060,
year = {2020},
author = {Garbett, D and Bisaria, A and Yang, C and McCarthy, DG and Hayer, A and Moerner, WE and Svitkina, TM and Meyer, T},
title = {T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4818},
pmid = {32968060},
issn = {2041-1723},
support = {F32 GM116328/GM/NIGMS NIH HHS/United States ; R01 GM095977/GM/NIGMS NIH HHS/United States ; R35 GM118067/GM/NIGMS NIH HHS/United States ; R35 GM127026/GM/NIGMS NIH HHS/United States ; },
mesh = {Actin Cytoskeleton/metabolism ; Actins/metabolism ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Line ; Cell Movement/*physiology ; Cell Surface Extensions/*metabolism ; Cytoskeleton/metabolism ; Extracellular Matrix/metabolism ; Gene Knockout Techniques ; Humans ; Kinetics ; Membrane Glycoproteins/genetics/*metabolism/ultrastructure ; Microfilament Proteins/genetics/*metabolism/ultrastructure ; Myosins/metabolism ; Pseudopodia/metabolism ; Receptor, EphB2 ; },
abstract = {Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.},
}
@article {pmid32966912,
year = {2020},
author = {Montaña, S and Vilacoba, E and Fernandez, JS and Traglia, GM and Sucari, A and Pennini, M and Iriarte, A and Centron, D and Melano, RG and Ramírez, MS},
title = {Genomic analysis of two Acinetobacter baumannii strains belonging to two different sequence types (ST172 and ST25).},
journal = {Journal of global antimicrobial resistance},
volume = {23},
number = {},
pages = {154-161},
doi = {10.1016/j.jgar.2020.09.006},
pmid = {32966912},
issn = {2213-7173},
abstract = {OBJECTIVES: Acinetobacter baumannii is an opportunistic nosocomial pathogen that is the main focus of attention in clinical settings owing to its intrinsic ability to persist in the hospital environment and its capacity to acquire determinants of resistance and virulence. Here we present the genomic sequencing, molecular characterisation and genomic comparison of two A. baumannii strains belonging to two different sequence types (STs), one sporadic and one widely distributed in our region.<br><br>METHODS: Whole-genome sequencing (WGS) of Ab42 and Ab376 was performed using Illumina MiSeq-I and the genomes were assembled with SPAdes. ARG-ANNOT, CARD-RGI, ISfinder, PHAST, PlasmidFinder, plasmidSPAdes and IslandViewer were used to analyse both genomes.<br><br>RESULTS: Genome analysis revealed that Ab42 belongs to ST172, an uncommon ST, whilst Ab376 belongs to ST25, a widely distributed ST. Molecular characterisation showed the presence of two antibiotic resistance genes in Ab42 and nine in Ab376. No insertion sequences were detected in Ab42, however 22 were detected in Ab376. Moreover, two prophages were found in Ab42 and three in Ab376. In addition, a CRISPR-cas type I-Fb and two plasmids, one of which harboured an AbGRI1-like island, were found in Ab376.<br><br>CONCLUSIONS: We present WGS analysis of twoA. baumannii strains belonging to two different STs. These findings allowed us to characterise a previously undescribed ST (ST172) and provide new insights to the widely studied ST25.},
}
@article {pmid32966770,
year = {2020},
author = {Wang, PY and Cripe, TP},
title = {Gene Editing Thumbs a Ride with Oncolytic Virotherapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {10},
pages = {2103-2104},
pmid = {32966770},
issn = {1525-0024},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; ErbB Receptors ; Gene Editing ; Genomics ; Humans ; *Neoplasms/genetics/therapy ; *Oncolytic Virotherapy ; *Oncolytic Viruses/genetics ; Thumb ; },
}
@article {pmid32966744,
year = {2020},
author = {Khan, P and Aufdembrink, LM and Engelhart, AE},
title = {Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings.},
journal = {ACS synthetic biology},
volume = {9},
number = {11},
pages = {2861-2880},
doi = {10.1021/acssynbio.0c00359},
pmid = {32966744},
issn = {2161-5063},
mesh = {COVID-19/*diagnosis/*epidemiology/prevention &amp; control/virology ; COVID-19 Testing/*methods ; CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; Polymerase Chain Reaction/methods ; SARS-CoV-2/*genetics/*immunology/isolation &amp; purification ; Self-Sustained Sequence Replication/methods ; Sensitivity and Specificity ; Serologic Tests/methods ; },
abstract = {The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 infection, past or present, in patients. We also discuss ongoing research on isothermal techniques, CRISPR-mediated detection assays, and point-of-care diagnostics that have potential for use in SARS-CoV-2 detection. Large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. The low-cost isothermal technologies described in this Review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks.},
}
@article {pmid32965719,
year = {2020},
author = {Rafia, C and Harly, C and Scotet, E},
title = {Beyond CAR T cells: Engineered Vγ9Vδ2 T cells to fight solid tumors.},
journal = {Immunological reviews},
volume = {298},
number = {1},
pages = {117-133},
doi = {10.1111/imr.12920},
pmid = {32965719},
issn = {1600-065X},
support = {PJA 20191209404//Association pour la Recherche contre le Cancer/ ; AOGO2019//Ligue Contre le Cancer/ ; //IMCHECK THERAPEUTICS/ ; PJA20191209404//Association pour la Recherche sur le Cancer/ ; FRM DEQ20170839118//Fondation pour la Recherche Médicale/ ; },
abstract = {Despite recent significant progress in cancer immunotherapies based on adoptive cell transfer(s)(ACT), the eradication of cancers still represents a major clinical challenge. In particular, the efficacy of current ACT-based therapies against solid tumors is dramatically reduced by physical barriers that prevent tumor infiltration of adoptively transferred effectors, and the tumor environment that suppress their anti-tumor functions. Novel immunotherapeutic strategies are thus needed to circumvent these issues. Human peripheral blood Vγ9Vδ2 T cells, a non-alloreactive innate-like T lymphocyte subset, recently proved to be a promising anti-tumor effector subset for ACT-based immunotherapies. Furthermore, new cell engineering tools that leverage the potential of CRISPR/Cas technology open astounding opportunities to optimize their anti-tumor effector functions. In this review, we present the current ACT strategies based on engineered T cells and their limitations. We then discuss the potential of engineered Vγ9Vδ2 T cell to overcome these limitations and improve ACT-based cancer immunotherapies.},
}
@article {pmid32960926,
year = {2020},
author = {McDiarmid, TA and Au, V and Moerman, DG and Rankin, CH},
title = {Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans.},
journal = {PloS one},
volume = {15},
number = {9},
pages = {e0238950},
pmid = {32960926},
issn = {1932-6203},
support = {R24 OD023041/OD/NIH HHS/United States ; PJT-148549//CIHR/Canada ; PJT-165947//CIHR/Canada ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; Caenorhabditis elegans Proteins/*genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Targeting/*methods ; Homozygote ; Phenotype ; RNA, Guide/genetics ; Toxins, Biological/*genetics/metabolism ; Transgenes ; },
abstract = {Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.},
}
@article {pmid32960291,
year = {2020},
author = {Mitsui, R and Yamada, R and Matsumoto, T and Yoshihara, S and Tokumoto, H and Ogino, H},
title = {Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficient D-lactic acid production.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {21},
pages = {9147-9158},
doi = {10.1007/s00253-020-10906-3},
pmid = {32960291},
issn = {1432-0614},
support = {JP18K14069//Japan Society for the Promotion of Science KAKENHI/ ; JP18KK0413//Japan Society for the Promotion of Science KAKENHI/ ; },
abstract = {Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct a D-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes, HXT7, HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH3, PGK1, GPM1, ENO2, and PYK2, and D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides were randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L of D-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L of D-LA from 100 g/L glucose with 20 g/L CaCO3 in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA. KEY POINTS: • Lactic acid (LA) tolerance of yeast was improved by genome evolution. • The transcription levels of 751 genes were changed under LA stress. • Rapid LA production with semi-neutralization was achieved by modifying glycolysis. • A versatile yeast strain construction method based on the CRISPR system was proposed.},
}
@article {pmid32960267,
year = {2020},
author = {Lin, J and Fuglsang, A and Kjeldsen, AL and Sun, K and Bhoobalan-Chitty, Y and Peng, X},
title = {DNA targeting by subtype I-D CRISPR-Cas shows type I and type III features.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {10470-10478},
pmid = {32960267},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/classification/*genetics ; CRISPR-Cas Systems/*genetics/immunology ; DNA/*genetics/immunology ; DNA Helicases/genetics ; DNA, Single-Stranded/genetics ; RNA, Bacterial/genetics ; RNA, Double-Stranded/genetics ; },
abstract = {Prokaryotic CRISPR-Cas immune systems are classified into six types based on their effector complexes which cleave dsDNA specifically (types I, II and V), ssRNA exclusively (type VI) or both ssRNA via a ruler mechanism and ssDNA unspecifically (type III). To date, no specific cleavage of ssDNA target has been reported for CRISPR-Cas. Here, we demonstrate dual dsDNA and ssDNA cleavage activities of a subtype I-D system which carries a type III Cas10-like large subunit, Cas10d. In addition to a specific dsDNA cleavage activity dependent on the HD domain of Cas10d, the helicase Cas3' and a compatible protospacer adjacent motif (PAM), the subtype I-D effector complex can cleave ssDNA that is complementary in sequence to the crRNA. Significantly, the ssDNA cleavage sites occur at 6-nt intervals and the cleavage is catalysed by the backbone subunit Csc2 (Cas7), similar to the periodic cleavage of ssRNA by the backbone subunit of type III effectors. The typical type I cleavage of dsDNA combined with the exceptional 6-nt spaced cleavage of ssDNA and the presence of a type III like large subunit provide strong evidence for the subtype I-D system being an evolutionary intermediate between type I and type III CRISPR-Cas systems.},
}
@article {pmid32959978,
year = {2020},
author = {Dai, Y},
title = {Chemical Translational Biology-Guided Molecular Diagnostics: The Front Line To Mediate the Current SARS-CoV-2 Pandemic.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {21},
number = {24},
pages = {3492-3494},
doi = {10.1002/cbic.202000518},
pmid = {32959978},
issn = {1439-7633},
mesh = {Biosensing Techniques/methods ; COVID-19/*diagnosis/epidemiology/virology ; CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/methods ; Pandemics ; Point-of-Care Testing ; RNA, Viral/*analysis ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2/genetics ; },
abstract = {The spread of severe respiratory syndrome coronavirus 2 (SARS-CoV-2) has disrupted our global society in unprecedented ways. The very front line in defense against this pandemic is molecular diagnosis, which is an exceptional representation of how chemical translational biology can benefit our lives. In this viewpoint, I emphasize the imperative demand for a simple and rapid point-of-care system in order to mediate the spread of COVID-19. I further describe how the interdisciplinary combination of chemistry and biology advances biosensing systems, which potentially lead to integrated and automated point-of-care systems capable of relieving the current pandemic.},
}
@article {pmid32959897,
year = {2020},
author = {Zhang, B},
title = {CRISPR/Cas gene therapy.},
journal = {Journal of cellular physiology},
volume = {},
number = {},
pages = {},
doi = {10.1002/jcp.30064},
pmid = {32959897},
issn = {1097-4652},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated enzyme (Cas) is a naturally occurring genome editing tool adopted from the prokaryotic adaptive immune defense system. Currently, CRISPR/Cas9-based genome editing has been becoming one of the most promising tools for treating human genetic diseases, including cardiovascular diseases, neuro-disorders, and cancers. As the quick modification of the CRISPR/Cas9 system, including delivery system, CRISPR/Cas9-based gene therapy has been extensively studied in preclinic and clinic treatments. CRISPR/Cas genome editing is also a robust tool to create animal genetic models for studying and treating human genetic disorders, particularly diseases associated with point mutations. However, significant challenges also remain before CRISPR/Cas technology can be routinely employed in the clinic for treating different genetic diseases, which include toxicity and immune response of treated cells to CRISPR/Cas component, highly throughput delivery method, and potential off-target impact. The off-target effect is one of the major concerns for CRISPR/Cas9 gene therapy, more research should be focused on limiting this impact by designing high specific gRNAs and using high specificity of Cas enzymes. Modifying the CRISPR/Cas9 delivery method not only targets a specific tissue/cell but also potentially limits the off-target impact.},
}
@article {pmid32959126,
year = {2020},
author = {Yu, J and Tu, L and Subburaj, S and Bae, S and Lee, GJ},
title = {Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.},
journal = {Plant cell reports},
volume = {},
number = {},
pages = {},
pmid = {32959126},
issn = {1432-203X},
support = {. PJ01319301//Next Generation BioGreen 21 Program/ ; },
abstract = {KEY MESSAGE: We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar 'Madness Midnight' has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer's field.},
}
@article {pmid32958931,
year = {2020},
author = {Xia, B and Amador, G and Viswanatha, R and Zirin, J and Mohr, SE and Perrimon, N},
title = {CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells.},
journal = {Nature protocols},
volume = {15},
number = {10},
pages = {3478-3498},
doi = {10.1038/s41596-020-0383-8},
pmid = {32958931},
issn = {1750-2799},
support = {R01 GM067761/GM/NIGMS NIH HHS/United States ; P41 GM132087/GM/NIGMS NIH HHS/United States ; R24 OD019847/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Alleles ; Animals ; Base Sequence/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drosophila/genetics ; Endonucleases/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Homozygote ; Polyploidy ; RNA, Guide/genetics ; },
abstract = {Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.},
}
@article {pmid32958637,
year = {2020},
author = {McWilliam, HEG and Mak, JYW and Awad, W and Zorkau, M and Cruz-Gomez, S and Lim, HJ and Yan, Y and Wormald, S and Dagley, LF and Eckle, SBG and Corbett, AJ and Liu, H and Li, S and Reddiex, SJJ and Mintern, JD and Liu, L and McCluskey, J and Rossjohn, J and Fairlie, DP and Villadangos, JA},
title = {Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {40},
pages = {24974-24985},
pmid = {32958637},
issn = {1091-6490},
mesh = {Antigen Presentation/genetics ; Antigens/genetics/immunology ; CRISPR-Cas Systems/genetics ; Endoplasmic Reticulum/*genetics ; Histocompatibility Antigens Class I/*genetics ; Humans ; Ligands ; Lymphocyte Activation/genetics ; Membrane Transport Proteins/genetics ; Metabolome/*genetics ; Minor Histocompatibility Antigens/*genetics ; Molecular Chaperones/genetics/immunology ; Mucosal-Associated Invariant T Cells/immunology ; *Proteomics ; Riboflavin/genetics ; },
abstract = {The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.},
}
@article {pmid32956422,
year = {2020},
author = {Krasnopolsky, S and Kuzmina, A and Taube, R},
title = {Genome-wide CRISPR knockout screen identifies ZNF304 as a silencer of HIV transcription that promotes viral latency.},
journal = {PLoS pathogens},
volume = {16},
number = {9},
pages = {e1008834},
pmid = {32956422},
issn = {1553-7374},
mesh = {CD4-Positive T-Lymphocytes/*metabolism/virology ; CRISPR-Cas Systems ; *Gene Expression Regulation, Viral ; Gene Knockout Techniques ; *Gene Silencing ; Genome-Wide Association Study ; HIV-1/*physiology ; Humans ; Jurkat Cells ; Promoter Regions, Genetic ; *Repressor Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; *Transcription, Genetic ; Tripartite Motif-Containing Protein 28/genetics/metabolism ; *Virus Latency ; },
abstract = {Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.},
}
@article {pmid32954022,
year = {2020},
author = {Abdullah, and Jiang, Z and Hong, X and Zhang, S and Yao, R and Xiao, Y},
title = {CRISPR base editing and prime editing: DSB and template-free editing systems for bacteria and plants.},
journal = {Synthetic and systems biotechnology},
volume = {5},
number = {4},
pages = {277-292},
pmid = {32954022},
issn = {2405-805X},
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) has been extensively exploited as a genetic tool for genome editing. The RNA guided Cas nucleases generate DNA double-strand break (DSB), triggering cellular repair systems mainly Non-homologous end-joining (NHEJ, imprecise repair) or Homology-directed repair (HDR, precise repair). However, DSB typically leads to unexpected DNA changes and lethality in some organisms. The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools. Hence, in this review, we focus on the non-DSB and template-free genome editing, i.e., base editing (BE) and prime editing (PE) in bacteria and plants. We first highlight the development of base and prime editors and summarize their studies in bacteria and plants. We then discuss current and future applications of BE/PE in synthetic biology, crop improvement, evolutionary engineering, and metabolic engineering. Lastly, we critically consider the challenges and prospects of BE/PE in PAM specificity, editing efficiency, off-targeting, sequence specification, and editing window.},
}
@article {pmid32951947,
year = {2020},
author = {Molla, KA and Qi, Y and Karmakar, S and Baig, MJ},
title = {Base Editing Landscape Extends to Perform Transversion Mutation.},
journal = {Trends in genetics : TIG},
volume = {36},
number = {12},
pages = {899-901},
doi = {10.1016/j.tig.2020.09.001},
pmid = {32951947},
issn = {0168-9525},
abstract = {Base editors have drawn considerable academic and industrial attention in recent years because of their ability to alter single DNA bases with precision. However, the existing cytosine and adenine base editors can only install transition mutations. Three recent studies (Kurt et al.,Zhao et al., and Chen et al.) expand the base editing toolbox by developing cytosine transversion base editors.},
}
@article {pmid32948757,
year = {2020},
author = {Ding, X and Yin, K and Li, Z and Lalla, RV and Ballesteros, E and Sfeir, MM and Liu, C},
title = {Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4711},
pmid = {32948757},
issn = {2041-1723},
support = {R01 CA214072/CA/NCI NIH HHS/United States ; R01 EB023607/EB/NIBIB NIH HHS/United States ; R21 TW010625/TW/FIC NIH HHS/United States ; },
mesh = {Betacoronavirus/genetics/*isolation &amp; purification ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/methods ; Coronavirus Infections/diagnosis/*virology ; Genes, Viral ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; Pneumonia, Viral/diagnosis/*virology ; Point-of-Care Systems ; RNA, Viral/analysis/genetics ; SARS-CoV-2 ; Sensitivity and Specificity ; Viral Proteins/analysis/genetics ; },
abstract = {The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.},
}
@article {pmid32946490,
year = {2020},
author = {Teixeira, LPR and Lopes, FEM and Antunes, ASLM and Alves, MS and Miranda, AM and Gaudencio Neto, S and Martins, LT and Moreira, ACOM and Tavares, KCS},
title = {Application of a cost-effective DNA extraction protocol for screening transgenic and CRISPR-edited primary goat cells.},
journal = {PloS one},
volume = {15},
number = {9},
pages = {e0239435},
pmid = {32946490},
issn = {1932-6203},
mesh = {Animals ; Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Cost-Benefit Analysis ; DNA/*genetics/*isolation &amp; purification ; Fibroblasts/cytology/metabolism ; *Gene Editing ; Goats ; Transgenes/*genetics ; },
abstract = {The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells-GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A-0%, protocol B-93%, protocol C-13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.},
}
@article {pmid32946000,
year = {2020},
author = {Gratacap, RL and Jin, YH and Mantsopoulou, M and Houston, RD},
title = {Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {22},
number = {5},
pages = {717-724},
pmid = {32946000},
issn = {1436-2236},
support = {BB/S004343/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/20002172/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/30002275/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002070/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.},
}
@article {pmid32941662,
year = {2020},
author = {Zhou Li, Y and Boisnard, S and Enache-Angoulvant, A and Fairhead, C},
title = {Genome editing in the yeast Nakaseomyces delphensis and description of its complete sexual cycle.},
journal = {Yeast (Chichester, England)},
volume = {},
number = {},
pages = {},
doi = {10.1002/yea.3522},
pmid = {32941662},
issn = {1097-0061},
abstract = {The environmental yeast Nakaseomyces delphensis is, phylogenetically, the closest known species to Candida glabrata, a major fungal pathogen of humans. C. glabrata is haploid and described as asexual, while N. delphensis is also haploid, but has been described as competent for mating and meiosis. Both genomes contain homologues of all the genes necessary for sexual reproduction and also the genes for Ho-dependent mating-type switching, like Saccharomyces cerevisiae. We first report the construction of genetically engineered strains of N. delphensis, including by CRISPR-Cas 9 gene editing. We also report the description of the sexual cycle of N. delphensis. We show that it undergoes Ho-dependent mating-type switching in culture and that deletion of the HO gene prevents such switching and allows maintenance of stable, separate, MATa and MATalpha haploid strains. Rare, genetically selected diploids can be obtained through mating of haploid strains, mutated or not for the HO gene. In contrast to HO/HO diploids, which behave as expected, Δho/Δho diploids exhibit unusual profiles in flow cytometry. Both types of diploids can produce recombined haploid cells, which grow like the original haploid-type strain. Our experiments thus allow the genetic manipulation of N. delphensis and the reconstruction, in the laboratory, of its entire life cycle.},
}
@article {pmid32941652,
year = {2020},
author = {Kim, DY and Moon, SB and Ko, JH and Kim, YS and Kim, D},
title = {Unbiased investigation of specificities of prime editing systems in human cells.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {10576-10589},
pmid = {32941652},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; *DNA Breaks, Single-Stranded ; Gene Editing/*methods ; Genome, Human/genetics ; Humans ; Whole Genome Sequencing ; },
abstract = {Prime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.},
}
@article {pmid32941625,
year = {2020},
author = {Nishimura, K and Yamada, R and Hagihara, S and Iwasaki, R and Uchida, N and Kamura, T and Takahashi, K and Torii, KU and Fukagawa, T},
title = {A super-sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {e108},
pmid = {32941625},
issn = {1362-4962},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Chickens ; Gene Knockout Techniques/*methods ; Humans ; Indoleacetic Acids/*chemistry ; Oryza/metabolism ; Plant Proteins/*genetics ; *Proteolysis ; },
abstract = {The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.},
}
@article {pmid32941607,
year = {2020},
author = {Kot, W and Olsen, NS and Nielsen, TK and Hutinet, G and de Crécy-Lagard, V and Cui, L and Dedon, PC and Carstens, AB and Moineau, S and Swairjo, MA and Hansen, LH},
title = {Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs.},
journal = {Nucleic acids research},
volume = {48},
number = {18},
pages = {10383-10396},
pmid = {32941607},
issn = {1362-4962},
mesh = {Bacteriophages/drug effects/*genetics ; Base Sequence/drug effects ; CRISPR-Cas Systems/genetics ; DNA/drug effects/*genetics ; DNA Restriction-Modification Enzymes/drug effects ; Escherichia coli/virology ; Gene Editing ; Guanine/analogs &amp; derivatives/pharmacology ; Humans ; Nanopore Sequencing ; Nucleotide Motifs/drug effects/*genetics ; Pyrimidinones/*pharmacology ; Pyrroles/*pharmacology ; Siphoviridae/genetics ; },
abstract = {In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.},
}
@article {pmid32939188,
year = {2020},
author = {Jolany Vangah, S and Katalani, C and Booneh, HA and Hajizade, A and Sijercic, A and Ahmadian, G},
title = {CRISPR-Based Diagnosis of Infectious and Noninfectious Diseases.},
journal = {Biological procedures online},
volume = {22},
number = {},
pages = {22},
pmid = {32939188},
issn = {1480-9222},
abstract = {Interest in CRISPR technology, an instrumental component of prokaryotic adaptive immunity which enables prokaryotes to detect any foreign DNA and then destroy it, has gained popularity among members of the scientific community. This is due to CRISPR's remarkable gene editing and cleaving abilities. While the application of CRISPR in human genome editing and diagnosis needs to be researched more fully, and any potential side effects or ambiguities resolved, CRISPR has already shown its capacity in an astonishing variety of applications related to genome editing and genetic engineering. One of its most currently relevant applications is in diagnosis of infectious and non-infectious diseases. Since its initial discovery, 6 types and 22 subtypes of CRISPR systems have been discovered and explored. Diagnostic CRISPR systems are most often derived from types II, V, and VI. Different types of CRISPR-Cas systems which have been identified in different microorganisms can target DNA (e.g. Cas9 and Cas12 enzymes) or RNA (e.g. Cas13 enzyme). Viral, bacterial, and non-infectious diseases such as cancer can all be diagnosed using the cleavage activity of CRISPR enzymes from the aforementioned types. Diagnostic tests using Cas12 and Cas13 enzymes have already been developed for detection of the emerging SARS-CoV-2 virus. Additionally, CRISPR diagnostic tests can be performed using simple reagents and paper-based lateral flow assays, which can potentially reduce laboratory and patient costs significantly. In this review, the classification of CRISPR-Cas systems as well as the basis of the CRISPR/Cas mechanisms of action will be presented. The application of these systems in medical diagnostics with emphasis on the diagnosis of COVID-19 will be discussed.},
}
@article {pmid32939090,
year = {2020},
author = {Zou, Y and Henry, WS and Ricq, EL and Graham, ET and Phadnis, VV and Maretich, P and Paradkar, S and Boehnke, N and Deik, AA and Reinhardt, F and Eaton, JK and Ferguson, B and Wang, W and Fairman, J and Keys, HR and Dančík, V and Clish, CB and Clemons, PA and Hammond, PT and Boyer, LA and Weinberg, RA and Schreiber, SL},
title = {Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.},
journal = {Nature},
volume = {585},
number = {7826},
pages = {603-608},
doi = {10.1038/s41586-020-2732-8},
pmid = {32939090},
issn = {1476-4687},
support = {U01 CA217848/CA/NCI NIH HHS/United States ; P01 CA080111/CA/NCI NIH HHS/United States ; R35 CA220487/CA/NCI NIH HHS/United States ; K99 CA248610/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Ethers/chemistry/*metabolism ; Female ; *Ferroptosis ; Gene Editing ; Humans ; Kidney Neoplasms/metabolism/pathology ; Lipid Peroxidation ; Male ; Mice ; Myocytes, Cardiac/cytology/metabolism ; Neurons/cytology/metabolism ; Ovarian Neoplasms/metabolism/pathology ; Peroxisomes/genetics/*metabolism ; Phospholipids/*chemistry/*metabolism ; },
abstract = {Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.},
}
@article {pmid32938767,
year = {2020},
author = {Liu, Y and Dai, L and Dong, J and Chen, C and Zhu, J and Rao, VB and Tao, P},
title = {Covalent Modifications of the Bacteriophage Genome Confer a Degree of Resistance to Bacterial CRISPR Systems.},
journal = {Journal of virology},
volume = {94},
number = {23},
pages = {},
pmid = {32938767},
issn = {1098-5514},
support = {R01 AI064389/AI/NIAID NIH HHS/United States ; U01 AI056443/AI/NIAID NIH HHS/United States ; R01 AI111538/AI/NIAID NIH HHS/United States ; U01 AI082086/AI/NIAID NIH HHS/United States ; T32 AI060549/AI/NIAID NIH HHS/United States ; R01 AI081726/AI/NIAID NIH HHS/United States ; },
abstract = {The interplay between defense and counterdefense systems of bacteria and bacteriophages has been driving the evolution of both organisms, leading to their great genetic diversity. Restriction-modification systems are well-studied defense mechanisms of bacteria, while phages have evolved covalent modifications as a counterdefense mechanism to protect their genomes against restriction. Here, we present evidence that these genome modifications might also have been selected to counter, broadly, the CRISPR-Cas systems, an adaptive bacterial defense mechanism. We found that the phage T4 genome modified by cytosine hydroxymethylation and glucosylation (ghmC) exhibits various degrees of resistance to the type V CRISPR-Cas12a system, producing orders of magnitude more progeny than the T4(C) mutant, which contains unmodified cytosines. Furthermore, the progeny accumulated CRISPR escape mutations, allowing rapid evolution of mutant phages under CRISPR pressure. A synergistic effect on phage restriction was observed when two CRISPR-Cas12a complexes were targeted to independent sites on the phage genome, another potential countermechanism by bacteria to more effectively defend themselves against modified phages. These studies suggest that the defense-counterdefense mechanisms exhibited by bacteria and phages, while affording protection against one another, also provide evolutionary benefits for both.IMPORTANCE Restriction-modification (R-M) and CRISPR-Cas systems are two well-known defense mechanisms of bacteria. Both recognize and cleave phage DNA at specific sites while protecting their own genomes. It is well accepted that T4 and other phages have evolved counterdefense mechanisms to protect their genomes from R-M cleavage by covalent modifications, such as the hydroxymethylation and glucosylation of cytosine. However, it is unclear whether such genome modifications also provide broad protection against the CRISPR-Cas systems. Our results suggest that genome modifications indeed afford resistance against CRISPR systems. However, the resistance is not complete, and it is also variable, allowing rapid evolution of mutant phages that escape CRISPR pressure. Bacteria in turn could target more than one site on the phage genome to more effectively restrict the infection of ghmC-modified phage. Such defense-counterdefense strategies seem to confer survival advantages to both the organisms, one of the possible reasons for their great diversity.},
}
@article {pmid32938718,
year = {2020},
author = {Szulc, B and Sosicka, P and Maszczak-Seneczko, D and Skurska, E and Shauchuk, A and Olczak, T and Freeze, HH and Olczak, M},
title = {Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {48},
pages = {16445-16463},
pmid = {32938718},
issn = {1083-351X},
support = {R01 DK099551/DK/NIDDK NIH HHS/United States ; },
abstract = {Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.},
}
@article {pmid32937129,
year = {2020},
author = {Zhao, R and Yang, Y and Zheng, F and Zeng, Z and Feng, W and Jin, X and Wang, J and Yang, K and Liang, YX and She, Q and Han, W},
title = {A Membrane-Associated DHH-DHHA1 Nuclease Degrades Type III CRISPR Second Messenger.},
journal = {Cell reports},
volume = {32},
number = {11},
pages = {108133},
doi = {10.1016/j.celrep.2020.108133},
pmid = {32937129},
issn = {2211-1247},
abstract = {Type III CRISPR-Cas systems initiate an intracellular signaling pathway to confer immunity. The signaling pathway includes synthesis of cyclic oligo-adenylate (cOA) and activation of the RNase activity of type III accessory ribonuclease Csm6/Csx1 by cOA. After the immune response, cOA should be cleared on time to avoid constant cellular RNA degradation. In this study, we find a metal-dependent cOA degradation activity in Sulfolobus islandicus. The activity is associated with the cell membrane and able to accelerate cOA clearance at a high cOA level. Further, we show that a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) rapidly cleaves cOA and deactivates Csx1 ribonuclease. The cOA degradation efficiency of MAD is much higher than the cellular ring nuclease. However, the subcellular organization may prevent it from degrading nascent cOA. Together, the data suggest that MAD acts as the second cOA degrader after the ring nuclease to remove diffused redundant cOA.},
}
@article {pmid32937062,
year = {2020},
author = {Joung, J and Ladha, A and Saito, M and Kim, NG and Woolley, AE and Segel, M and Barretto, RPJ and Ranu, A and Macrae, RK and Faure, G and Ioannidi, EI and Krajeski, RN and Bruneau, R and Huang, MW and Yu, XG and Li, JZ and Walker, BD and Hung, DT and Greninger, AL and Jerome, KR and Gootenberg, JS and Abudayyeh, OO and Zhang, F},
title = {Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing.},
journal = {The New England journal of medicine},
volume = {383},
number = {15},
pages = {1492-1494},
doi = {10.1056/NEJMc2026172},
pmid = {32937062},
issn = {1533-4406},
support = {DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; 1DP1-HL141201/NH/NIH HHS/United States ; 1R01- MH110049/NH/NIH HHS/United States ; },
mesh = {Betacoronavirus/genetics/*isolation &amp; purification ; COVID-19 ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/*diagnosis ; Humans ; Pandemics ; Pneumonia, Viral/*diagnosis ; RNA, Viral/*analysis ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2 ; Sensitivity and Specificity ; },
}
@article {pmid32936799,
year = {2020},
author = {Fiaux, PC and Chen, HV and Chen, PB and Chen, AR and McVicker, G},
title = {Discovering functional sequences with RELICS, an analysis method for CRISPR screens.},
journal = {PLoS computational biology},
volume = {16},
number = {9},
pages = {e1008194},
pmid = {32936799},
issn = {1553-7358},
support = {P30 CA014195/CA/NCI NIH HHS/United States ; R01 DK122607/DK/NIDDK NIH HHS/United States ; R01 AI107027/AI/NIAID NIH HHS/United States ; },
mesh = {Bayes Theorem ; CRISPR-Cas Systems/*genetics ; Genomics/*methods ; Humans ; Jurkat Cells ; RNA, Guide/genetics ; Sequence Analysis, DNA/*methods ; *Software ; },
abstract = {CRISPR screens are a powerful technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By targeting non-coding sequences for perturbation, CRISPR screens have the potential to systematically discover novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from CRISPR screens. RELICS specifically addresses many of the challenges of non-coding CRISPR screens such as the unknown locations of functional sequences, overdispersion in the observed single guide RNA counts, and the need to combine information across multiple pools in an experiment. RELICS outperforms existing methods with higher precision, higher recall, and finer-resolution predictions on simulated datasets. We apply RELICS to published CRISPR interference and CRISPR activation screens to predict and experimentally validate novel regulatory sequences that are missed by other analysis methods. In summary, RELICS is a powerful new analysis method for CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy.},
}
@article {pmid32930628,
year = {2020},
author = {Gholizadeh, P and Aghazadeh, M and Ghotaslou, R and Ahangarzadeh Rezaee, M and Pirzadeh, T and Köse, Ş and Ganbarov, K and Yousefi, M and Kafil, HS},
title = {CRISPR-cas system in the acquisition of virulence genes in dental-root canal and hospital-acquired isolates of Enterococcus faecalis.},
journal = {Virulence},
volume = {11},
number = {1},
pages = {1257-1267},
pmid = {32930628},
issn = {2150-5608},
abstract = {Enterococcus faecalis is one of the important causative agents of nosocomial and life-threatening infections in human. Several studies have demonstrated that the presence of CRISPR-cas is associated with antibiotic susceptibility and lack of virulence traits. In this study, we aimed to assess the phenotypic and genotypic virulence determinants in relation to CRISPR elements from the dental-root canals and hospital-acquired isolates of E. faecalis. Eighty-eight hospital-acquired and 73 dental-root canal isolates of E. faecalis were assessed in this study. Phenotypic screening of the isolates included biofilm formation, and gelatinase and hemolysis activities. Genotypical screening using PCR was further used to evaluate the presence of CRISPR elements and different virulence-associated genes such as efaA, esp, cylA, hyl, gelE, ace, ebpR, and asa1. Biofilm formation, gelatinase, and hemolysis activities were detected in 93.8%, 29.2%, and 19.2% of the isolates, respectively. The most prevalent virulence-associated gene was ace, which was followed by efaA, whereas cylA was the least identified. The presence of CRISPR1-cas, orphan CRISPR2, and CRISPR3-cas was determined in 13%, 55.3%, and 17.4% of the isolates, respectively. CRISPR elements were significantly more prevalent in the dental-root canal isolates. An inverse significant correlation was found between CRISPR-cas loci, esp, and gelE, while direct correlations were observed in the case of cylA, hyl, gelE (among CRISPR-loci 1 and 3), asa1, ace, biofilm formation, and hemolysis activity. Findings, therefore, indicate that CRISPR-cas might prevent the acquisition of some respective pathogenicity factors in some isolates, though not all; so selective forces could not influence pathogenic traits. Abbreviations: BHI: brain-heart infusion agar; CRISPRs: Clustered regularly interspaced short palindromic repeats; Esp: Cell wall-associated protein; ENT: ear-nose-throat; ICU: intensive care units; OD: optical densities; PCR: polymerase chain reaction; SDS: sodium dodecyl sulfate; UTI: urinary tract infection.},
}
@article {pmid32929070,
year = {2020},
author = {Li, Y and Weng, Y and Bai, D and Jia, Y and Liu, Y and Zhang, Y and Kou, X and Zhao, Y and Ruan, J and Chen, J and Yin, J and Wang, H and Teng, X and Wang, Z and Liu, W and Gao, S},
title = {Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4593},
pmid = {32929070},
issn = {2041-1723},
mesh = {*Alleles ; Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Nucleus/*genetics ; Disease Models, Animal ; Dwarfism/genetics ; Embryo Loss/genetics ; Female ; *Gene Editing ; Gene Targeting ; Genes, Dominant ; Genomic Imprinting ; Heterozygote ; Mice, Inbred C57BL ; Mice, Knockout ; *Mitochondrial Replacement Therapy ; Mutation ; Reproducibility of Results ; Time Factors ; },
abstract = {Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.},
}
@article {pmid32929034,
year = {2020},
author = {Champer, J and Yang, E and Lee, E and Liu, J and Clark, AG and Messer, PW},
title = {A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {39},
pages = {24377-24383},
pmid = {32929034},
issn = {1091-6490},
support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Drosophila Proteins/*genetics ; Drosophila melanogaster/*genetics/physiology ; Female ; Gene Editing ; Germ Cells/cytology ; Male ; Models, Genetic ; Pedigree ; RNA, Guide/genetics ; },
abstract = {Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. Here, we present a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire populations.},
}
@article {pmid32926383,
year = {2021},
author = {Shin, HR and Kweon, J and Kim, Y},
title = {Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {185-193},
doi = {10.1007/978-1-0716-0687-2_10},
pmid = {32926383},
issn = {1940-6029},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein has emerged as a genome engineering tool for various organisms. Known as the CRISPR-Cas system, Cas endonucleases such as Cas9 and Cas12a (also known as Cpf1) and guide RNA (gRNA) complexes recognize and cleave the target DNA, allowing for targeted gene manipulation. Along with the Cas protein engineering, gRNA engineering has broadened the applications of the CRISPR-Cas system. Recently, we have developed fusion guide RNAs (fgRNAs) for orthogonal gene manipulation using Cas9 and Cas12a. Here, we describe the methods for designing and generating fgRNAs-expression constructs to achieve multiplex genome editing and gene manipulation in human cells.},
}
@article {pmid32926375,
year = {2021},
author = {Hwang, GH and Kim, JS and Bae, S},
title = {Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {23-33},
doi = {10.1007/978-1-0716-0687-2_2},
pmid = {32926375},
issn = {1940-6029},
abstract = {The CRISPR-Cas system facilitates highly efficient genome editing; thus, it has been applied in many research fields such as biological science, medicine, and gene therapy. However, CRISPR nucleases can cleave off-target sites as well as on-target sites, causing unwanted mutations. Furthermore, after CRISPR treatments are delivered into cells or organisms, it is important to estimate the resulting mutation rates and to determine the patterns of mutations, but these tasks can be difficult. To address these issues, we have developed a tool for identifying potential off-target sites (Cas-OFFinder), a tool for designing CRISPR targets (Cas-Designer), and an assessment tool (Cas-Analyzer). These programs are all implemented on our website so that researchers can easily design CRISPR guide RNAs and assess the resulting mutations by simply clicking on the appropriate buttons; no login process is required.},
}
@article {pmid32926374,
year = {2021},
author = {Heigwer, F and Boutros, M},
title = {Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2162},
number = {},
pages = {3-22},
doi = {10.1007/978-1-0716-0687-2_1},
pmid = {32926374},
issn = {1940-6029},
abstract = {CRISPR/Cas-based genome editing in any biological application requires the evaluation of suitable genomic target sites to design efficient reagents. Considerations for the design of short guide (sg) RNAs include the assessment of possible off-target activities, the prediction of on-target efficacies and mutational outcome. Manual design of sgRNAs taking into account these parameters, however, remains a difficult task. Thus, computational tools to design sgRNA reagents from small scale to genome-wide libraries have been developed that assist during all steps of the design process. Here, we will describe practical guidance for the sgRNA design process using the web-based tool E-CRISP used in the design of individual sgRNAs. E-CRISP (www.e-crisp.org) has been the first web-based sgRNA design tool and uniquely features simple, yet efficient, scoring schemes in combination with fast evaluation and simple usage. We will also discuss the installation of a dockerized version of CRISPR Library Designer (CLD) that can be deployed locally or in the cloud to support the end-to-end design of sgRNA libraries for more than 50 different organisms. CLD was built upon E-CRISP to further increase the scope of sgRNA design to more experimental modalities (CRISPRa/i, Cas12a, all possible protospacer adjacency motifs) offering the same flexibility as E-CRISP, plus the scalability through local and cloud installation. Together, these tools facilities the design of small and large-scale CRISPR/Cas experiments.},
}
@article {pmid32926267,
year = {2020},
author = {Song, X and Zhang, XY and Xiong, ZQ and Liu, XX and Xia, YJ and Wang, SJ and Ai, LZ},
title = {CRISPR-Cas-mediated gene editing in lactic acid bacteria.},
journal = {Molecular biology reports},
volume = {47},
number = {10},
pages = {8133-8144},
doi = {10.1007/s11033-020-05820-w},
pmid = {32926267},
issn = {1573-4978},
support = {2019-02-08-00-07-F01152//Shanghai Agriculture Applied Technology Development Program/ ; 31871757//Natural Science Foundation of China/ ; 18DZ2200200//Shanghai Technical Standard Program/ ; 19DZ2281100//Shanghai Engineering Research Center of food microbiology program/ ; },
abstract = {The high efficiency, convenience and diversity of clustered regular interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are driving a technological revolution in the gene editing of lactic acid bacteria (LAB). Cas-RNA cassettes have been adopted as tools to perform gene deletion, insertion and point mutation in several species of LAB. In this article, we describe the basic mechanisms of the CRISPR-Cas system, and the current gene editing methods available, focusing on the CRISPR-Cas models developed for LAB. We also compare the different types of CRISPR-Cas-based genomic manipulations classified according to the different Cas proteins and the type of recombineering, and discuss the rapidly evolving landscape of CRISPR-Cas application in LAB.},
}
@article {pmid32920019,
year = {2020},
author = {Riedl, A and Gruber, S and Ruzsics, Z},
title = {Novel conditional plasmids regulated by chemical switches provide versatile tools for genetic engineering in Escherichia coli.},
journal = {Plasmid},
volume = {111},
number = {},
pages = {102531},
doi = {10.1016/j.plasmid.2020.102531},
pmid = {32920019},
issn = {1095-9890},
abstract = {Engineering bacterial genomes or foreign DNA cloned as bacterial artificial chromosomes (BACs) relies on usage of helper plasmids, which deliver the desired tools transiently into the bacteria to be modified. After the anticipated action is completed the helper plasmids need to be cured. To make this efficient, plasmids are used that are maintained by conditional amplicons or carry a counter-selection marker. Here, we describe new conditional plasmids that can be maintained or cured by using chemical induction or repression. Our method is based on the dependency of plasmids carrying ori6Kγ origin of replication on the presence of protein Π. Ori6Kγ based plasmids are tightly regulated conditional constructs, but they require usually special E. coli strains to operate. To avoid this, we placed the Π protein expression under the control of a co-expressed conditional repressor. Regulating the maintenance of plasmids with administration or removal of chemicals is fully compatible with any other conditional amplicons applied to date. Here, we describe methods for inducing sites specific recombination of BACs as an example. However, the same strategy might be used to construct appropriate helper plasmids for any other transient components of genome editing methodologies such as λred recombinases or CRISPR/Cas components.},
}
@article {pmid32918585,
year = {2020},
author = {Feng, S and Hu, L and Zhang, Q and Zhang, F and Du, J and Liang, G and Li, A and Song, G and Liu, Y},
title = {CRISPR/Cas technology promotes the various application of Dunaliella salina system.},
journal = {Applied microbiology and biotechnology},
volume = {104},
number = {20},
pages = {8621-8630},
doi = {10.1007/s00253-020-10892-6},
pmid = {32918585},
issn = {1432-0614},
support = {No. U1804112, No. 31571289//the National Natural Science Foundation of China/ ; },
abstract = {Dunaliella salina (D. salina) has been widely applied in various fields because of its inherent advantages, such as the study of halotolerant mechanism, wastewater treatment, recombinant proteins expression, biofuel production, preparation of natural materials, and others. However, owing to the existence of low yield or in the laboratory exploration stage, D. salina system has been greatly restricted for practical production of various components. In past decade, significant progresses have been achieved for research of D. salina in these fields. Among them, D. salina as a novel expression system demonstrated a bright prospect, especially for large-scale production of foreign proteins, like the vaccines, antibodies, and other therapeutic proteins. Due to the low efficiency, application of traditional regulation tools is also greatly limited for exploration of D. salina system. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system offers a precise editing tool to overcome the obstacles of D. salina system. This review not only comprehensively summarizes the recent progresses of D. salina in domain of gene engineering but also gives a deep analysis of problems and deficiencies in different fields of D. salina. Moreover, further prospects of CRISPR/Cas system and its significant challenges have been discussed in various aspects of D. salina. It provides a great referencing value for speeding up the maturity of D. salina system, and also supplies practical guiding significance to expand the new application fields for D. salina. KEY POINTS: • The review provides recent research progresses of various applications of D. salina. • The problems and deficiencies in different fields of D. salina were deeply analyzed. • The further prospects of CRISPR/Cas technology in D. salina system were predicted. • CRISPR/Cas system will promote the new application fields and maturity for D. salina.},
}
@article {pmid32917893,
year = {2020},
author = {Franklin, JM and Ghosh, RP and Shi, Q and Reddick, MP and Liphardt, JT},
title = {Concerted localization-resets precede YAP-dependent transcription.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4581},
pmid = {32917893},
issn = {2041-1723},
support = {U01 EB021237/EB/NIBIB NIH HHS/United States ; U54 CA143836/CA/NCI NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/genetics/*metabolism ; CRISPR-Cas Systems ; Calcium/metabolism ; Cell Line, Tumor ; Cell Nucleus/metabolism ; Cytoplasm/metabolism ; Gene Expression Regulation ; Gene Knock-In Techniques ; HEK293 Cells ; Humans ; Mechanotransduction, Cellular/physiology ; Oncogenes/genetics ; Transcription Factors/genetics/*metabolism ; },
abstract = {Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus (&quot;localization-resets&quot;) activates YAP target genes. These &quot;resets&quot; are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.},
}
@article {pmid32915222,
year = {2019},
author = {Mills, P},
title = {Preimplantation genome editing: CCR5 in China.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {6},
pages = {695-700},
doi = {10.1042/ETLS20190114},
pmid = {32915222},
issn = {2397-8554},
mesh = {CRISPR-Cas Systems/*genetics ; China ; Gene Editing ; Genetic Therapy ; HIV Infections/*prevention &amp; control/*therapy ; Humans ; Receptors, CCR5/*genetics ; Targeted Gene Repair ; },
abstract = {Part of the criticism of the one reported case of human preimplantation genome editing (PGE) turned on the inadequacy of the purpose for which it was undertaken (inherent immunity to HIV) and its target (the CCR5 gene). The discussion of CCR5 in this context reveals the different values that inform the idea of acceptable uses of PGE and of the conditions of responsible biomedical innovation among the scientist responsible and his critics. While the use of PGE for any indication remains unacceptable (or, at the very least, premature), neither position offers a satisfactory response to this prospective biotechnology.},
}
@article {pmid32915215,
year = {2019},
author = {Iacomussi, S},
title = {Regulating genome editing technologies: a comparison of expert recommendations in the U.K. and in the U.S.A.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {6},
pages = {701-705},
doi = {10.1042/ETLS20180101},
pmid = {32915215},
issn = {2397-8554},
mesh = {CRISPR-Cas Systems ; England ; Gene Editing/*legislation &amp; jurisprudence/trends ; Genetic Therapy ; Government Regulation ; Humans ; Public Policy ; United States ; },
abstract = {The present paper aims to inform the bioethical debate on the regulation of human genome editing technologies with a specific focus on the role of scientific experts and their interactions with the general public in the formulation of policy. It reviews and compares two of the major contributions to this debate in the U.K. and in the U.S.A., comparing expert approaches towards regulation on genome editing technologies. The results of this analysis offer important lessons that should be appreciated in building an international regulatory framework. On the basis of these results, I conclude that the experts should embrace a socially responsible approach and encourage active public engagement.},
}
@article {pmid32915213,
year = {2019},
author = {Bryant, JA},
title = {From bacterial battles to CRISPR crops; progress towards agricultural applications of genome editing.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {6},
pages = {687-693},
doi = {10.1042/ETLS20190065},
pmid = {32915213},
issn = {2397-8554},
mesh = {Agriculture ; CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; Disease Resistance/genetics ; Gene Editing/*methods ; Genetic Engineering ; Genome, Plant ; Humans ; Plants, Genetically Modified/*genetics ; },
abstract = {Genome editing is the precise alteration of DNA in living cells by the cutting or removal of specific sequences, sometimes followed by insertion of new sequences at the cut site. CRISPR-Cas9 has become firmly established as the genome-editing method of choice, replacing the systems that had been developed and in use since the early 1990s. The CRISPR-Cas9 system has been developed from a mechanism used in prokaryotes as a defence against bacteriophage but actually functions in cells of all types of organisms. It is widely used in research as a gene knockout and editing tool; applications in veterinary medicine (such as increased resistance to disease) and human medicine (such as correction of disease-causing mutations) are under development. In agriculture and horticulture, the potential for various aspects of crop improvement is very large. Selected aspects of this potential are presented here, with particular focus on crop quality and disease resistance. The article ends with a brief discussion of the regulatory 'environment' in the USA and the EU.},
}
@article {pmid32913194,
year = {2020},
author = {Watry, HL and Feliciano, CM and Gjoni, K and Takahashi, G and Miyaoka, Y and Conklin, BR and Judge, LM},
title = {Rapid, precise quantification of large DNA excisions and inversions by ddPCR.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {14896},
pmid = {32913194},
issn = {2045-2322},
support = {R01 EY028249/EY/NEI NIH HHS/United States ; R01 HL130533/HL/NHLBI NIH HHS/United States ; R01 HL135358/HL/NHLBI NIH HHS/United States ; P01 HL146366/HL/NHLBI NIH HHS/United States ; U01 EB029374/EB/NIBIB NIH HHS/United States ; },
abstract = {The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (> 20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.},
}
@article {pmid32913081,
year = {2020},
author = {Cohen, J},
title = {Narrow path charted for editing genes of human embryos.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6509},
pages = {1283},
doi = {10.1126/science.369.6509.1283},
pmid = {32913081},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; Embryo Research/*ethics ; Gene Editing/*ethics ; Genetic Diseases, Inborn/genetics/therapy ; Humans ; Practice Guidelines as Topic ; Reproduction/genetics ; },
}
@article {pmid32912679,
year = {2020},
author = {Pattharaprachayakul, N and Lee, M and Incharoensakdi, A and Woo, HM},
title = {Current understanding of the cyanobacterial CRISPR-Cas systems and development of the synthetic CRISPR-Cas systems for cyanobacteria.},
journal = {Enzyme and microbial technology},
volume = {140},
number = {},
pages = {109619},
doi = {10.1016/j.enzmictec.2020.109619},
pmid = {32912679},
issn = {1879-0909},
abstract = {Cyanobacteria are photosynthetic microorganisms that are capable of converting CO2 to value-added chemicals. Engineering of cyanobacteria with synthetic biology tools, including the CRISPR-Cas system, has allowed an opportunity for biological CO2 utilization. Here, we described natural CRISPR-Cas systems for understanding cyanobacterial genomics and synthetic CRISPR-Cas systems for metabolic engineering applications. The natural CRISPR-Cas systems in cyanobacteria have been identified as Class 1, with type I and III, and some Class 2, with type V, as an adaptive immune system against viral invasion. As synthetic tools, CRISPR-Cas9 and -Cas12a have been successfully established in cyanobacteria to delete a target gene without a selection marker. Deactivated Cas9 and Cas12a have also been used to repress genes for metabolic engineering. In addition, a perspective on how advanced CRISPR-Cas systems and a pool of the guide RNAs can be advantageous for precise genome engineering and understanding of unknown functions was discussed for advanced engineering of cyanobacteria.},
}
@article {pmid32912325,
year = {2020},
author = {Gabr, H and El Ghamrawy, MK and Almaeen, AH and Abdelhafiz, AS and Hassan, AOS and El Sissy, MH},
title = {CRISPR-mediated gene modification of hematopoietic stem cells with beta-thalassemia IVS-1-110 mutation.},
journal = {Stem cell research &amp; therapy},
volume = {11},
number = {1},
pages = {390},
pmid = {32912325},
issn = {1757-6512},
abstract = {BACKGROUND: β-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation.<br><br>METHODS AND RESULTS: We successfully isolated CD34+ cells from peripheral blood of β-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34+ cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin.<br><br>CONCLUSION: CRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of β-thalassemia.},
}
@article {pmid32912206,
year = {2020},
author = {Schleidgen, S and Dederer, HG and Sgodda, S and Cravcisin, S and Lüneburg, L and Cantz, T and Heinemann, T},
title = {Human germline editing in the era of CRISPR-Cas: risk and uncertainty, inter-generational responsibility, therapeutic legitimacy.},
journal = {BMC medical ethics},
volume = {21},
number = {1},
pages = {87},
pmid = {32912206},
issn = {1472-6939},
support = {01GP1616A-C//Bundesministerium für Bildung und Forschung/International ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) technology may allow for efficient and highly targeted gene editing in single-cell embryos. This possibility brings human germline editing into the focus of ethical and legal debates again.<br><br>MAIN BODY: Against this background, we explore essential ethical and legal questions of interventions into the human germline by means of CRISPR-Cas: How should issues of risk and uncertainty be handled? What responsibilities arise regarding future generations? Under which conditions can germline editing measures be therapeutically legitimized? For this purpose, we refer to a scenario anticipating potential further development in CRISPR-Cas technology implying improved accuracy and exclusion of germline transmission to future generations. We show that, if certain concepts regarding germline editing are clarified, under such conditions a categorical prohibition of one-generation germline editing of single-cell embryos appears not to be ethically or legally justifiable.<br><br>CONCLUSION: These findings are important prerequisites for the international debate on the ethical and legal justification of germline interventions in the human embryo as well as for the harmonization of international legal standards.},
}
@article {pmid32907944,
year = {2020},
author = {Lin, SC and Qu, L and Ettayebi, K and Crawford, SE and Blutt, SE and Robertson, MJ and Zeng, XL and Tenge, VR and Ayyar, BV and Karandikar, UC and Yu, X and Coarfa, C and Atmar, RL and Ramani, S and Estes, MK},
title = {Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {38},
pages = {23782-23793},
pmid = {32907944},
issn = {1091-6490},
support = {U19 AI144297/AI/NIAID NIH HHS/United States ; U19 AI116497/AI/NIAID NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; HHSN272201700081C/AI/NIAID NIH HHS/United States ; P30 ES030285/ES/NIEHS NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; T32 DK007664/DK/NIDDK NIH HHS/United States ; P01 AI057788/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Caliciviridae Infections/immunology/virology ; Host-Pathogen Interactions/*immunology ; Humans ; *Interferons/genetics/metabolism ; *Intestines/immunology/virology ; Models, Biological ; *Norovirus/genetics/immunology/pathogenicity ; Organoids/immunology/virology ; Sequence Analysis, RNA ; Transcriptome/genetics ; Virus Replication ; },
abstract = {Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2 An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.},
}
@article {pmid32904687,
year = {2020},
author = {Nemudryi, A and Nemudraia, A and Wiegand, T and Surya, K and Buyukyoruk, M and Cicha, C and Vanderwood, KK and Wilkinson, R and Wiedenheft, B},
title = {Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater.},
journal = {Cell reports. Medicine},
volume = {1},
number = {6},
pages = {100098},
pmid = {32904687},
issn = {2666-3791},
abstract = {SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here, we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 74-day time course. We show that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results. In addition, we determine a nearly complete (98.5%) SARS-CoV-2 genome sequence from wastewater and use phylogenetic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for genotyping viral strains circulating in a community.},
}
@article {pmid32903830,
year = {2020},
author = {Song, S and Wood, TK},
title = {A Primary Physiological Role of Toxin/Antitoxin Systems Is Phage Inhibition.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {1895},
pmid = {32903830},
issn = {1664-302X},
abstract = {Toxin/antitoxin (TA) systems are present in most prokaryote genomes. Toxins are almost exclusively proteins that reduce metabolism (but do not cause cell death), and antitoxins are either RNA or proteins that counteract the toxin or the RNA that encodes it. Although TA systems clearly stabilize mobile genetic elements, after four decades of research, the physiological roles of chromosomal TA systems are less clear. For example, recent reports have challenged the notion of TA systems as stress-response elements, including a role in creating the dormant state known as persistence. Here, we present evidence that a primary physiological role of chromosomally encoded TA systems is phage inhibition, a role that is also played by some plasmid-based TA systems. This includes results that show some CRISPR-Cas system elements are derived from TA systems and that some CRISPR-Cas systems mimic the host growth inhibition invoked by TA systems to inhibit phage propagation.},
}
@article {pmid32903507,
year = {2020},
author = {Rittiner, JE and Moncalvo, M and Chiba-Falek, O and Kantor, B},
title = {Gene-Editing Technologies Paired With Viral Vectors for Translational Research Into Neurodegenerative Diseases.},
journal = {Frontiers in molecular neuroscience},
volume = {13},
number = {},
pages = {148},
pmid = {32903507},
issn = {1662-5099},
abstract = {Diseases of the central nervous system (CNS) have historically been among the most difficult to treat using conventional pharmacological approaches. This is due to a confluence of factors, including the limited regenerative capacity and overall complexity of the brain, problems associated with repeated drug administration, and difficulties delivering drugs across the blood-brain barrier (BBB). Viral-mediated gene transfer represents an attractive alternative for the delivery of therapeutic cargo to the nervous system. Crucially, it usually requires only a single injection, whether that be a gene replacement strategy for an inherited disorder or the delivery of a genome- or epigenome-modifying construct for treatment of CNS diseases and disorders. It is thus understandable that considerable effort has been put towards the development of improved vector systems for gene transfer into the CNS. Different viral vectors are of course tailored to their specific applications, but they generally should share several key properties. The ideal viral vector incorporates a high-packaging capacity, efficient gene transfer paired with robust and sustained expression, lack of oncogenicity, toxicity and pathogenicity, and scalable manufacturing for clinical applications. In this review, we will devote attention to viral vectors derived from human immunodeficiency virus type 1 (lentiviral vectors; LVs) and adeno-associated virus (AAVs). The high interest in these viral delivery systems vectors is due to: (i) robust delivery and long-lasting expression; (ii) efficient transduction into postmitotic cells, including the brain; (iii) low immunogenicity and toxicity; and (iv) compatibility with advanced manufacturing techniques. Here, we will outline basic aspects of LV and AAV biology, particularly focusing on approaches and techniques aiming to enhance viral safety. We will also allocate a significant portion of this review to the development and use of LVs and AAVs for delivery into the CNS, with a focus on the genome and epigenome-editing tools based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) and the development of novel strategies for the treatment of neurodegenerative diseases (NDDs).},
}
@article {pmid32903441,
year = {2020},
author = {Newire, E and Aydin, A and Juma, S and Enne, VI and Roberts, AP},
title = {Identification of a Type IV-A CRISPR-Cas System Located Exclusively on IncHI1B/IncFIB Plasmids in Enterobacteriaceae.},
journal = {Frontiers in microbiology},
volume = {11},
number = {},
pages = {1937},
pmid = {32903441},
issn = {1664-302X},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are diverse immune systems found in many prokaryotic genomes that target invading foreign DNA such as bacteriophages and plasmids. There are multiple types of CRISPR with arguably the most enigmatic being Type IV. During an investigation of CRISPR carriage in clinical, multi-drug resistant, Klebsiella pneumoniae, a Type IV-A3 CRISPR-Cas system was detected on plasmids from two K. pneumoniae isolates from Egypt (isolated in 2002-2003) and a single K. pneumoniae isolate from the United Kingdom (isolated in 2017). Sequence analysis of all other genomes available in GenBank revealed that this CRISPR-Cas system was present on 28 other plasmids from various Enterobacteriaceae hosts and was never found on a bacterial chromosome. This system is exclusively located on IncHI1B/IncFIB plasmids and is associated with multiple putative transposable elements. Expression of the cas loci was confirmed in the available clinical isolates by RT-PCR. In all cases, the CRISPR-Cas system has a single CRISPR array (CRISPR1) upstream of the cas loci which has several, conserved, spacers which, amongst things, match regions within conjugal transfer genes of IncFIIK/IncFIB(K) plasmids. Our results reveal a Type IV-A3 CRISPR-Cas system exclusively located on IncHI1B/IncFIB plasmids in Enterobacteriaceae that is likely to be able to target IncFIIK/IncFIB(K) plasmids presumably facilitating intracellular, inter-plasmid competition.},
}
@article {pmid32900994,
year = {2020},
author = {Crone, MA and Priestman, M and Ciechonska, M and Jensen, K and Sharp, DJ and Anand, A and Randell, P and Storch, M and Freemont, PS},
title = {A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4464},
doi = {10.1038/s41467-020-18130-3},
pmid = {32900994},
issn = {2041-1723},
support = {NIHR-RP-011-048/DH_/Department of Health/United Kingdom ; },
mesh = {Betacoronavirus/*genetics/isolation &amp; purification ; Biological Assay ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; Clinical Laboratory Techniques/instrumentation/*methods/standards ; Coronavirus Infections/*diagnosis/*virology ; Humans ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Pandemics ; Pneumonia, Viral/*diagnosis/*virology ; RNA, Viral/analysis/genetics/isolation &amp; purification ; Real-Time Polymerase Chain Reaction ; SARS-CoV-2 ; Sensitivity and Specificity ; },
abstract = {The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.},
}
@article {pmid32896179,
year = {2020},
author = {Chauhan, N and Soni, S and Gupta, A and Jain, U},
title = {New and developing diagnostic platforms for COVID-19: A systematic review.},
journal = {Expert review of molecular diagnostics},
volume = {20},
number = {9},
pages = {971-983},
doi = {10.1080/14737159.2020.1816466},
pmid = {32896179},
issn = {1744-8352},
mesh = {Betacoronavirus/*genetics ; Biosensing Techniques ; COVID-19 ; COVID-19 Testing ; CRISPR-Cas Systems ; *Clinical Laboratory Techniques ; Coronavirus Infections/*diagnosis/diagnostic imaging ; Expert Testimony ; Humans ; Pandemics ; Pneumonia, Viral/*diagnosis ; Reagent Kits, Diagnostic ; Reverse Transcriptase Polymerase Chain Reaction/methods ; SARS-CoV-2 ; Tomography, X-Ray Computed/methods ; Ultrasonography/methods ; },
abstract = {INTRODUCTION: The starting months of 2020 witnessed a global pandemic of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. The first case of Coronavirus Disease 2019 (COVID-19) was reported in December, 2019 in Wuhan, China and millions of cases and thousands of deaths were reported within five months. Currently, reverse transcription-polymerase chain reaction (RT-PCR) and computed tomography (CT) scanning are clinically prescribed for COVID-19 detection across the globe.<br><br>AREAS COVERED: This systematic review is focused on currently used diagnostic methods for COVID-19 detection and their future prospects. Online searches on Google Scholar, PubMed and online resources were conducted on the period of year 2017 to mid-2020. Studies investigating laboratory examinations, radiographical analysis, and potential sensors for COVID-19 detection were included. Along with this, the current status of commercially available kits for SARS-CoV-2 coronavirus detection is discussed.<br><br>EXPERT OPINION: The search has identified the potential applications of nucleic acid technology, diagnostics radiology examinations, and in-vitro diagnostic kits in detection of COVID-19 infections. Despite having their own limitations of each technology, the emerging diagnostic technologies for COVID-19 detection along with undergoing clinical trials are summarized suggesting more collaborations and funding are required for fast track clinical trials.},
}
@article {pmid32895556,
year = {2020},
author = {Zhang, H and Li, Z and Xiao, R and Chang, L},
title = {Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease.},
journal = {Nature structural &amp; molecular biology},
volume = {27},
number = {11},
pages = {1069-1076},
doi = {10.1038/s41594-020-0499-0},
pmid = {32895556},
issn = {1545-9985},
abstract = {Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially be used for genome editing with high specificity. To elucidate its mechanisms for target recognition and cleavage, we determined cryo-EM structures of Cas12i in multiple functional states. Cas12i pre-orders a seven-nucleotide seed sequence of the crRNA for target recognition and undergoes a two-step activation through crRNA-DNA hybridization. Formation of 14 base pairs activates the nickase activity, and 28-bp hybridization promotes cleavage of the target strand. The atomic structures and mechanistic insights gained should facilitate the manipulation of Cas12i for genome editing applications.},
}
@article {pmid32895531,
year = {2020},
author = {Baker, M},
title = {When antibodies mislead: the quest for validation.},
journal = {Nature},
volume = {585},
number = {7824},
pages = {313-314},
doi = {10.1038/d41586-020-02549-1},
pmid = {32895531},
issn = {1476-4687},
mesh = {Antibodies/*immunology ; Antibody Specificity/*immunology ; *Artifacts ; CRISPR-Cas Systems ; Fluorescent Antibody Technique/standards ; Gene Knockout Techniques ; Immunoblotting/standards ; Immunoprecipitation/standards ; Indicators and Reagents/*standards ; Reproducibility of Results ; *Research Design ; *Validation Studies as Topic ; },
}
@article {pmid32893479,
year = {2020},
author = {Saba, LM and Hoffman, PL and Homanics, GE and Mahaffey, S and Daulatabad, SV and Janga, SC and Tabakoff, B},
title = {A long non-coding RNA (Lrap) modulates brain gene expression and levels of alcohol consumption in rats.},
journal = {Genes, brain, and behavior},
volume = {},
number = {},
pages = {e12698},
doi = {10.1111/gbb.12698},
pmid = {32893479},
issn = {1601-183X},
support = {//Banbury Fund/ ; U01AA020889//Role of noncoding RNA in Alcohol Action/ ; R24AA013162//The Heritable Transcriptome and Alcoholism/ ; R01GM123314//Mapping RNA Protein Interaction Networks in the Human Genome/ ; R37AA010422//Ethanol Mechanisms in GABAAR gene targeted mice/ ; P30DA044223//Overall NIDA Core &quot;Center of Excellence&quot; in Transcriptomics, Systems Genetics and the Addictome/ ; U01 AA020889/AA/NIAAA NIH HHS/United States ; },
abstract = {LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The &quot;hub gene&quot; of this module, Lrap (Long non-coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of &quot;Response to Ethanol&quot; emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non-coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype.},
}
@article {pmid32891884,
year = {2020},
author = {Schenke, D and Cai, D},
title = {Applications of CRISPR/Cas to Improve Crop Disease Resistance: Beyond Inactivation of Susceptibility Factors.},
journal = {iScience},
volume = {23},
number = {9},
pages = {101478},
pmid = {32891884},
issn = {2589-0042},
abstract = {Current crop production systems are prone to increasing pathogen pressure. Fundamental understanding of molecular plant-pathogen interactions, the availability of crop and pathogen genomic information, as well as emerging genome editing permits a novel approach for breeding of crop disease resistance. We describe here strategies to identify new targets for resistance breeding with focus on interruption of the compatible plant-pathogen interaction by CRISPR/Cas-mediated genome editing. Basically, crop genome editing can be applied in several ways to achieve this goal. The most common approach focuses on the &quot;simple&quot; knockout by non-homologous end joining repair of plant susceptibility factors required for efficient host colonization. However, genome re-writing via homology-directed repair or base editing can also prevent host manipulation by changing the targets of pathogen-derived effectors or molecules beyond recognition, which also decreases plant susceptibility. We conclude that genome editing by CRISPR/Cas will become increasingly indispensable to generate in relatively short time beneficial resistance traits in crops to meet upcoming challenges.},
}
@article {pmid32891680,
year = {2020},
author = {Boucher, P and Cui, X and Curiel, DT},
title = {Adenoviral vectors for in vivo delivery of CRISPR-Cas gene editors.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {327},
number = {},
pages = {788-800},
doi = {10.1016/j.jconrel.2020.09.003},
pmid = {32891680},
issn = {1873-4995},
abstract = {Harnessing the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system for genome editing in eukaryotes has revolutionized basic biomedical research and translational sciences. The ability to create targeted alterations of the genome through this easy to design system has presented unprecedented opportunities to treat inherited disorders and other diseases such as cancer through gene therapy. A major hurdle is the lack of an efficient and safe in vivo delivery system, limiting most of the current gene therapy efforts to ex vivo editing of extracted cells. Here we discuss the unique features of adenoviral vectors that enable tissue specific and efficient delivery of the CRISPR-Cas machinery for in vivo genome editing.},
}
@article {pmid32890931,
year = {2020},
author = {Wang, DX and Wang, J and Du, YC and Ma, JY and Wang, SY and Tang, AN and Kong, DM},
title = {CRISPR/Cas12a-based dual amplified biosensing system for sensitive and rapid detection of polynucleotide kinase/phosphatase.},
journal = {Biosensors &amp; bioelectronics},
volume = {168},
number = {},
pages = {112556},
doi = {10.1016/j.bios.2020.112556},
pmid = {32890931},
issn = {1873-4235},
abstract = {We reported a CRISPR/Cas-based dual amplified sensing strategy for rapid, sensitive and selective detection of polynucleotide kinase/phosphatase (PNKP), a DNA damage repair-related biological enzyme. In this strategy, a PNKP-triggered nicking enzyme-mediated strand displacement amplification reaction was introduced to enrich the activator DNA strands for CRISPR/Cas. Such an isothermal DNA amplification step, together with subsequent activated CRISPR/Cas-catalyzed cleavage of fluorescent-labeled short-stranded DNA probes, enable synergetic signal amplification for sensitive PNKP detection. The proposed strategy showed a wide linear detection range (more than 3 orders of magnitude ranging from 1× 10-5 to 2.5 × 10-2 U/mL T4 PNKP) and a detection limit as low as 3.3 × 10-6 U/mL. It was successfully used for the PNKP activity detection in cell extracts with high fidelity and displayed great potential for enzyme inhibitor screening and inhibitory capability evaluation. This work broadens the applications of CRISPR/Cas12a-based sensors to biological enzymes and provides a way to improve the sensitivity by introducing an isothermal signal amplification step. Such an isothermal DNA amplification-CRISPR/Cas-combined biosensor design concept might expand CRISPR/Cas-based sensing systems and promote their applications in various fields such as disease diagnosis and drug screening.},
}
@article {pmid32888436,
year = {2020},
author = {Bacman, SR and Moraes, CT},
title = {Mitochondrial DNA Base Editing: Good Editing Things Still Come in Small Packages.},
journal = {Molecular cell},
volume = {79},
number = {5},
pages = {708-709},
doi = {10.1016/j.molcel.2020.08.009},
pmid = {32888436},
issn = {1097-4164},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Cytidine Deaminase ; *DNA, Mitochondrial ; Mitochondria/genetics ; },
abstract = {The collaborative work of two HHMI groups, one at the University of Washington and the other at the Broad Institute of MIT and Harvard, led to the development of a novel molecular tool to edit single bases in the mtDNA (Mok et al., 2020).},
}
@article {pmid32888434,
year = {2020},
author = {Collins, SP and Beisel, CL},
title = {Your Base Editor Might Be Flirting with Single (Stranded) DNA: Faithful On-Target CRISPR Base Editing without Promiscuous Deamination.},
journal = {Molecular cell},
volume = {79},
number = {5},
pages = {703-704},
doi = {10.1016/j.molcel.2020.07.030},
pmid = {32888434},
issn = {1097-4164},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosine ; DNA, Single-Stranded ; Deamination ; *Gene Editing ; },
abstract = {Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.},
}
@article {pmid32888044,
year = {2020},
author = {Liu, T and Zhu, L and Zhu, Z and Jiang, L},
title = {Genome Sequence Analysis of Clostridium tyrobutyricum, a Promising Microbial Host for Human Health and Industrial Applications.},
journal = {Current microbiology},
volume = {77},
number = {11},
pages = {3685-3694},
doi = {10.1007/s00284-020-02175-0},
pmid = {32888044},
issn = {1432-0991},
support = {2017YFC1600404//National Key R&amp;D Program of China/ ; 31922070//National Natural Science Foundation of China/ ; BK20171461//Natural Science Foundation of Jiangsu Province/ ; BK20180038//Natural Science Foundation of Jiangsu Province/ ; KYCX19_0868//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; 2012AA021705//National High-tech Research and Development Program/ ; },
abstract = {Clostridium tyrobutyricum is a promising microbial host for the anaerobic production of bio-based chemicals, especially butyric acid. At the same time, it also has great potential as a probiotic for the production of short-chain fatty acids in the intestines. However, due to the insufficient knowledge of the genetic characteristics of this organism, there has been little progress in its genetic engineering to date. Here, we present the complete genome sequence of C. tyrobutyricum L319, which consists of a circular chromosome and a plasmid with a G + C content of 31.69%, encompassing approximately 3.09 Mb with 3052 protein-coding genes. Functional gene annotation revealed better results than previous studies based on KEGG pathway classification. Furthermore, we obtained detailed functional characterization of 93 genes previously annotated as putative proteins. Genomic analysis revealed that this organism contains multiple genes encoding enzymes involved in the CRISPR-Cas systems, substrate utilization, isopeptide and ester bonds, transcriptional regulation, and oxidative stress. The safety evaluation at genetic level indicated that this organism does not possess transferable resistance genes, invasive defensive pathogenicity factors, or harmful enzymes. The genome sequence data analyzed in this study will be available for further research and will facilitate the further understanding and development of potential applications of C. tyrobutyricum.},
}
@article {pmid32887975,
year = {2020},
author = {Roman Azcona, MS and Fang, Y and Carusillo, A and Cathomen, T and Mussolino, C},
title = {A versatile reporter system for multiplexed screening of effective epigenome editors.},
journal = {Nature protocols},
volume = {15},
number = {10},
pages = {3410-3440},
doi = {10.1038/s41596-020-0380-y},
pmid = {32887975},
issn = {1750-2799},
mesh = {CRISPR-Cas Systems ; Cell Line ; DNA/genetics ; DNA Methylation/genetics ; Epigenesis, Genetic/genetics ; Epigenome/*genetics ; Epigenomics/*methods ; Gene Editing/*methods ; Genes, Reporter/genetics ; Genome/genetics ; HEK293 Cells ; Humans ; },
abstract = {The formation and function of highly specialized cells and tissues in a multicellular organism from a single genome are enabled through differential spatiotemporal access to the information contained in the genomic DNA. The epigenome plays an essential role in how DNA information can be accessed, and in the last decade the link between epigenetic aberrations and pathologies has become increasingly clear. Methods to precisely modify the epigenome are hence attracting interest as potential novel therapeutics. We recently described a platform, designer epigenome modifier (DEM), capable of precisely editing the epigenome of a cell to control the expression of selected genes. Here, we provide a detailed protocol to streamline the process of identifying DEMs that efficiently and selectively bind to their intended target site and inactivate expression of the target gene. Further, we describe the procedure to simultaneously regulate the expression of up to three genes in a multiplexed fashion. The protocol is divided into four stages that guide the user through the generation of the multicolor reporter cell line and its use for selecting functional DEMs. The duration of the whole procedure described varies from ~6 weeks when using a single reporter up to 13 weeks for fine-tuning the multiplex epigenome editing abilities of selected DEMs using three reporters. Given the great interest in epigenome editing in various fields of biomedical research, this protocol will help scientists to explore these novel technologies for their research.},
}
@article {pmid32887885,
year = {2020},
author = {Schmidt, C and Fransz, P and Rönspies, M and Dreissig, S and Fuchs, J and Heckmann, S and Houben, A and Puchta, H},
title = {Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {4418},
pmid = {32887885},
issn = {2041-1723},
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems ; Chromosome Inversion ; *Chromosomes, Plant ; Crossing Over, Genetic ; Genetic Engineering/*methods ; Plant Breeding/methods ; Plants ; *Recombination, Genetic ; },
abstract = {Chromosomal inversions are recurrent rearrangements that occur between different plant isolates or cultivars. Such inversions may underlie reproductive isolation in evolution and represent a major obstacle for classical breeding as no crossovers can be observed between inverted sequences on homologous chromosomes. The heterochromatic knob (hk4S) on chromosome 4 is the most well-known inversion of Arabidopsis. If a knob carrying accession such as Col-0 is crossed with a knob-less accession such as Ler-1, crossovers cannot be recovered within the inverted region. Our work shows that by egg-cell specific expression of the Cas9 nuclease from Staphylococcus aureus, a targeted reversal of the 1.1 Mb long hk4S-inversion can be achieved. By crossing Col-0 harbouring the rearranged chromosome 4 with Ler-1, meiotic crossovers can be restored into a region with previously no detectable genetic exchange. The strategy of somatic chromosome engineering for breaking genetic linkage has huge potential for application in plant breeding.},
}
@article {pmid32887353,
year = {2020},
author = {Patrulea, V and Borchard, G and Jordan, O},
title = {An Update on Antimicrobial Peptides (AMPs) and Their Delivery Strategies for Wound Infections.},
journal = {Pharmaceutics},
volume = {12},
number = {9},
pages = {},
pmid = {32887353},
issn = {1999-4923},
abstract = {Bacterial infections occur when wound healing fails to reach the final stage of healing, which is usually hindered by the presence of different pathogens. Different topical antimicrobial agents are used to inhibit bacterial growth due to antibiotic failure in reaching the infected site, which is accompanied very often by increased drug resistance and other side effects. In this review, we focus on antimicrobial peptides (AMPs), especially those with a high potential of efficacy against multidrug-resistant and biofilm-forming bacteria and fungi present in wound infections. Currently, different AMPs undergo preclinical and clinical phase to combat infection-related diseases. AMP dendrimers (AMPDs) have been mentioned as potent microbial agents. Various AMP delivery strategies that are used to combat infection and modulate the healing rate-such as polymers, scaffolds, films and wound dressings, and organic and inorganic nanoparticles-have been discussed as well. New technologies such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (CRISPR-Cas) are taken into consideration as potential future tools for AMP delivery in skin therapy.},
}
@article {pmid32886694,
year = {2020},
author = {Madaha, EL and Mienie, C and Gonsu, HK and Bughe, RN and Fonkoua, MC and Mbacham, WF and Alayande, KA and Bezuidenhout, CC and Ateba, CN},
title = {Whole-genome sequence of multi-drug resistant Pseudomonas aeruginosa strains UY1PSABAL and UY1PSABAL2 isolated from human broncho-alveolar lavage, Yaoundé, Cameroon.},
journal = {PloS one},
volume = {15},
number = {9},
pages = {e0238390},
pmid = {32886694},
issn = {1932-6203},
mesh = {Base Composition ; Cameroon ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Multiple/*genetics ; Genome, Bacterial/genetics ; Humans ; Phylogeny ; Prophages/genetics ; Pseudomonas aeruginosa/*genetics/isolation &amp; purification ; Sequence Analysis, DNA ; Virulence/genetics ; Whole Genome Sequencing/methods ; },
abstract = {Pseudomonas aeruginosa has been implicated in a wide range of post-operation wound and lung infections. A wide range of acquired resistance and virulence markers indicate surviving strategy of P. aeruginosa. Complete-genome analysis has been identified as efficient approach towards understanding the pathogenicity of this organism. This study was designed to sequence the entire genome of P. aeruginosa UY1PSABAL and UY1PSABAL2; determine drug-resistance profiles and virulence factors of the isolates; assess factors that contribute toward stability of the genomes; and thereafter determine evolutionary relationships between the strains and other isolates from similar sources. The genomes of the MDR P. aeruginosa UY1PSABAL and UY1PSABAL2 were sequenced on the Illumina Miseq platform. The raw sequenced reads were assessed for quality using FastQC v.0.11.5 and filtered for low quality reads and adapter regions using Trimmomatic v.0.36. The de novo genome assembly was made with SPAdes v.3.13 and annotated using Prokka v.2.1.1 annotation pipeline; Rapid Annotation using Subsytems Technology (RAST) server v.2.0; and PATRIC annotation tool v.3.6.2. Antimicrobial resistance genes and virulence determinants were searched through the functional annotation data generated from Prokka, RAST and PATRIC annotation pipelines; In addition to ResFinder and Comprehensive Antibiotic Resistance Database (CARD) which were employed to determine resistance genes. The PHAge Search Tool Enhanced Release (PHASTER) web server was used for the rapid identification and annotation of prophage sequences within bacterial genome. Predictive secondary metabolites were identified with AntiSMASH v.5.0. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and cas genes regions were also investigated with the CRISPRone and CRISPRFinder server. The genome sizes of 7.0 and 6.4 Mb were determined for UY1PSABAL and UY1PSABAL2 strains with G+C contents of 66.1% and 66.48% respectively. β-lactamines resistance genes blaPAO, aminoglycoside phosphorylating enzymes genes aph(3')-IIb, fosfomycine resistance gene fosA, vancomycin vanW and tetracycline tetA were among identified resistance genes harboured in both isolates. UY1PSABAL bore additional aph(6)-Id, aph(3'')-Ib, ciprofloxacin-modifying enzyme crpP and ribosomal methylation enzyme rmtB. Both isolates were found harbouring virulence markers such as flagella and type IV pili; and also present various type III secretion systems such as exoA, exoS, exoU, exoT. Secondary metabolites such as pyochelin and pyoverdine with iron uptake activity were found within the genomes as well as quorum-sensing systems, and various fragments for prophages and insertion sequences. Only the UY1PSABAL2 contains CRISPR-Cas system. The phylogeny revealed a very close evolutionary relationship between UY1PSABAL and the similar strain isolated from Malaysia; the same trend was observed between UY1PSABAL2 and the strain from Chinese origin. Complete analyses of the entire genomes provide a wide range of information towards understanding pathogenicity of the pathogens in question.},
}
@article {pmid32884147,
year = {2020},
author = {Moghadam, F and LeGraw, R and Velazquez, JJ and Yeo, NC and Xu, C and Park, J and Chavez, A and Ebrahimkhani, MR and Kiani, S},
title = {Synthetic immunomodulation with a CRISPR super-repressor in vivo.},
journal = {Nature cell biology},
volume = {22},
number = {9},
pages = {1143-1154},
pmid = {32884147},
issn = {1476-4679},
support = {R01 HL141805/HL/NHLBI NIH HHS/United States ; U01 EB029372/EB/NIBIB NIH HHS/United States ; R01 EB024562/EB/NIBIB NIH HHS/United States ; R01 EB028532/EB/NIBIB NIH HHS/United States ; P30 DK120531/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/*immunology ; Gene Editing/methods ; HEK293 Cells ; Humans ; Immunomodulation/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Myeloid Differentiation Factor 88/immunology ; Proprotein Convertase 9 ; RNA, Guide/immunology ; Receptors, Cell Surface/immunology ; },
abstract = {Transient modulation of the genes involved in immunity, without exerting a permanent change in the DNA code, can be an effective strategy to modulate the course of many inflammatory conditions. CRISPR-Cas9 technology represents a promising platform for achieving this goal. Truncation of guide RNA (gRNA) from the 5' end enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multifunctionality of CRISPR. Here, we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo. In this repressor system, two transcriptional repressors-heterochromatin protein 1 (HP1a) and Krüppel-associated box (KRAB)-are fused to the MS2 coat protein and subsequently recruited by gRNA aptamer binding to a nuclease competent CRISPR complex containing truncated gRNAs. With the enhanced repressor, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 (Myd88) gene in vitro and in vivo. We demonstrate that this strategy can efficiently downregulate Myd88 expression in lung, blood and bone marrow of Cas9 transgenic mice that receive systemic injection of adeno-associated virus (AAV)2/1-carrying truncated gRNAs targeting Myd88 and the MS2-HP1a-KRAB cassette. This downregulation is accompanied by changes in downstream signalling elements such as TNF-α and ICAM-1. Myd88 repression leads to a decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and this strategy modulates the IgG response against AAV cargos. It improves the efficiency of a subsequent AAV9/CRISPR treatment for repression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a gene that, when repressed, can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicaemia in both Cas9 transgenic and C57BL/6J mice. When delivered by nanoparticles, this repressor can serve as a therapeutic modality to influence the course of septicaemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate the host immune response against AAV-mediated gene therapy and influence the course of septicaemia. The ability to control Myd88 transcript levels using a CRISPR-based synthetic repressor can be an effective strategy for AAV-based CRISPR therapies, as this pathway serves as a key node in the induction of humoral immunity against AAV serotypes.},
}
@article {pmid32884115,
year = {2020},
author = {Ray, U and Raghavan, SC},
title = {Modulation of DNA double-strand break repair as a strategy to improve precise genome editing.},
journal = {Oncogene},
volume = {39},
number = {41},
pages = {6393-6405},
doi = {10.1038/s41388-020-01445-2},
pmid = {32884115},
issn = {1476-5594},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*drug effects/genetics ; DNA Repair Enzymes/*antagonists &amp; inhibitors/metabolism ; DNA-Binding Proteins/antagonists &amp; inhibitors/metabolism ; Gene Editing/*methods ; Gene Knockdown Techniques ; Homologous Recombination/*drug effects/genetics ; Humans ; Models, Animal ; Oligonucleotides, Antisense/pharmacology ; Pyrimidines/pharmacology ; Schiff Bases/pharmacology ; },
abstract = {In the present day, it is possible to incorporate targeted mutations or replace a gene using genome editing techniques such as customisable CRISPR/Cas9 system. Although induction of DNA double-strand breaks (DSBs) by genome editing tools can be repaired by both non-homologous end joining (NHEJ) and homologous recombination (HR), the skewness of the former pathway in human and other mammals normally result in imprecise repair. Scientists working at the crossroads of DNA repair and genome editing have devised new strategies for using a specific pathway to their advantage. Refinement in the efficiency of precise gene editing was witnessed upon downregulation of NHEJ by knockdown or using small molecule inhibitors on one hand, and upregulation of HR proteins and addition of HR stimulators, other hand. The exploitation of cell cycle phase differences together with appropriate donor DNA length/sequence and small molecules has provided further improvement in precise genome editing. The present article reviews the mechanisms of improving the efficiency of precise genome editing in several model organisms and in clinics.},
}
@article {pmid32883881,
year = {2020},
author = {Ewen-Campen, B and Perrimon, N},
title = {Expanding the horizons of genome editing in the fruit fly with Cas12a.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {39},
pages = {24019-24021},
pmid = {32883881},
issn = {1091-6490},
support = {R24 OD026435/OD/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; *CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Drosophila/metabolism ; *Gene Editing ; RNA, Guide ; },
}
@article {pmid32883277,
year = {2020},
author = {Liu, Z and Dong, H and Cui, Y and Cong, L and Zhang, D},
title = {Application of different types of CRISPR/Cas-based systems in bacteria.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {172},
pmid = {32883277},
issn = {1475-2859},
support = {NSFC 31800086//National Natural Science Foundation of China/ ; },
abstract = {As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12a-mediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.},
}
@article {pmid32881707,
year = {2020},
author = {Ryan, D and Prezza, G and Westermann, AJ},
title = {An RNA-centric view on gut Bacteroidetes.},
journal = {Biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1515/hsz-2020-0230},
pmid = {32881707},
issn = {1437-4315},
abstract = {Bacteria employ noncoding RNAs to maintain cellular physiology, adapt global gene expression to fluctuating environments, sense nutrients, coordinate their interaction with companion microbes and host cells, and protect themselves against bacteriophages. While bacterial RNA research has made fundamental contributions to biomedicine and biotechnology, the bulk of our knowledge of RNA biology stems from the study of a handful of aerobic model species. In comparison, RNA research is lagging in many medically relevant obligate anaerobic species, in particular the numerous commensal bacteria comprising our gut microbiota. This review presents a guide to RNA-based regulatory mechanisms in the phylum Bacteroidetes, focusing on the most abundant bacterial genus in the human gut, Bacteroides spp. This includes recent case reports on riboswitches, an mRNA leader, cis- and trans-encoded small RNAs (sRNAs) in Bacteroides spp., and a survey of CRISPR-Cas systems across Bacteroidetes. Recent work from our laboratory now suggests the existence of hundreds of noncoding RNA candidates in Bacteroides thetaiotaomicron, the emerging model organism for functional microbiota research. Based on these collective observations, we predict mechanistic and functional commonalities and differences between Bacteroides sRNAs and those of other model bacteria, and outline open questions and tools needed to boost Bacteroidetes RNA research.},
}
@article {pmid32877304,
year = {2021},
author = {Fiaz, S and Wang, X and Younas, A and Alharthi, B and Riaz, A and Ali, H},
title = {Apomixis and strategies to induce apomixis to preserve hybrid vigor for multiple generations.},
journal = {GM crops &amp; food},
volume = {12},
number = {1},
pages = {57-70},
pmid = {32877304},
issn = {2164-5701},
mesh = {*Apomixis ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing ; Hybrid Vigor ; Seeds ; },
abstract = {Hybrid seeds of several important crops with supreme qualities including yield, biotic and abiotic stress tolerance have been cultivated for decades. Thus far, a major challenge with hybrid seeds is that they do not have the ability to produce plants with the same qualities over subsequent generations. Apomixis, an asexual mode of reproduction by avoiding meiosis, exists naturally in flowering plants, and ultimately leads to seed production. Apomixis has the potential to preserve hybrid vigor for multiple generations in economically important plant genotypes. The evolution and genetics of asexual seed production are unclear, and much more effort will be required to determine the genetic architecture of this phenomenon. To fix hybrid vigor, synthetic apomixis has been suggested. The development of MiMe (mitosis instead of meiosis) genotypes has been utilized for clonal gamete production. However, the identification and parental origin of genes responsible for synthetic apomixis are little known and need further clarification. Genome modifications utilizing genome editing technologies (GETs), such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (cas), a reverse genetics tool, have paved the way toward the utilization of emerging technologies in plant molecular biology. Over the last decade, several genes in important crops have been successfully edited. The vast availability of GETs has made functional genomics studies easy to conduct in crops important for food security. Disruption in the expression of genes specific to egg cell MATRILINEAL (MTL) through the CRISPR/Cas genome editing system promotes the induction of haploid seed, whereas triple knockout of the Baby Boom (BBM) genes BBM1, BBM2, and BBM3 cause embryo arrest and abortion, which can be fully rescued by male-transmitted BBM1. The establishment of synthetic apomixis by engineering the MiMe genotype by genome editing of BBM1 expression or disruption of MTL leads to clonal seed production and heritability for multiple generations. In the present review, we discuss current developments related to the use of CRISPR/Cas technology in plants and the possibility of promoting apomixis in crops to preserve hybrid vigor. In addition, genetics, evolution, epigenetic modifications, and strategies for MiMe genotype development are discussed in detail.},
}
@article {pmid32875201,
year = {2020},
author = {Tyagi, S and Kesiraju, K and Saakre, M and Rathinam, M and Raman, V and Pattanayak, D and Sreevathsa, R},
title = {Genome Editing for Resistance to Insect Pests: An Emerging Tool for Crop Improvement.},
journal = {ACS omega},
volume = {5},
number = {33},
pages = {20674-20683},
pmid = {32875201},
issn = {2470-1343},
abstract = {Plants are challenged incessantly by several biotic and abiotic stresses during their entire growth period. As with other biotic stress factors, insect pests have also posed serious concerns related to yield losses due to which agricultural productivity is at stake. In plants, trait modification for crop improvement was initiated with breeding approaches followed by genetic engineering. However, stringent regulatory policies for risk assessment and lack of social acceptance for genetically modified crops worldwide have incited researchers toward alternate strategies. Genome engineering or genome editing has emerged as a new breeding technique with the ability to edit the genomes of plants, animals, microbes, and human beings. Several gene editing strategies are being executed with continuous emergence of variants. The scientific community has unraveled the utility of various editing tools from endonucleases to CRISPR/Cas in several aspects related to plant growth, development, and mitigation of stresses. The categorical focus on the development of tools and techniques including designing of binary vectors to facilitate ease in genome engineering are being pursued. Through this Review, we embark upon the conglomeration of various genome editing strategies that can be and are being used to design insect pest resistance in plants. Case studies and novel crop-based approaches that reiterate the successful use of these tools in insects as well as in plants are highlighted. Further, the Review also provides implications for the requirement of a specific regulatory framework and risk assessment of the edited crops. Genome editing toward insect pest management is here to stay, provided uncompromising efforts are made toward the identification of amiable target genes.},
}
@article {pmid32873648,
year = {2020},
author = {Davis, L and Khoo, KJ and Zhang, Y and Maizels, N},
title = {POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {37},
pages = {22900-22909},
pmid = {32873648},
issn = {1091-6490},
support = {R01 CA183967/CA/NCI NIH HHS/United States ; R21 CA190675/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *DNA Breaks, Double-Stranded ; DNA Breaks, Single-Stranded ; DNA End-Joining Repair ; DNA Ligases/genetics/metabolism ; DNA-Directed DNA Polymerase/genetics/*metabolism ; Heterozygote ; Humans ; Loss of Heterozygosity ; Mutation ; Recombination, Genetic ; *Recombinational DNA Repair ; },
abstract = {Interhomolog recombination (IHR) occurs spontaneously in somatic human cells at frequencies that are low but sufficient to ameliorate some genetic diseases caused by heterozygous mutations or autosomal dominant mutations. Here we demonstrate that DNA nicks or double-strand breaks (DSBs) targeted by CRISPR-Cas9 to both homologs can stimulate IHR and associated copy-neutral loss of heterozygosity (cnLOH) in human cells. The frequency of IHR is 10-fold lower at nicks than at DSBs, but cnLOH is evident in a greater fraction of recombinants. IHR at DSBs occurs predominantly via reciprocal end joining. At DSBs, depletion of POLQ caused a dramatic increase in IHR and in the fraction of recombinants exhibiting cnLOH, suggesting that POLQ promotes end joining in cis, which limits breaks available for recombination in trans These results define conditions that may produce cnLOH as a mutagenic signature in cancer and may, conversely, promote therapeutic correction of both compound heterozygous and dominant negative mutations associated with genetic disease.},
}
@article {pmid32872311,
year = {2020},
author = {Kantor, A and McClements, ME and MacLaren, RE},
title = {CRISPR-Cas9 DNA Base-Editing and Prime-Editing.},
journal = {International journal of molecular sciences},
volume = {21},
number = {17},
pages = {},
pmid = {32872311},
issn = {1422-0067},
abstract = {Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.},
}
@article {pmid32871045,
year = {2020},
author = {Ren, J and Yu, D and Fu, R and An, P and Sun, R and Wang, Z and Guo, R and Li, H and Zhang, Y and Li, Z and Yang, YG and Li, W and Hai, T and Hu, Z},
title = {IL2RG-deficient minipigs generated via CRISPR/Cas9 technology support the growth of human melanoma-derived tumours.},
journal = {Cell proliferation},
volume = {53},
number = {10},
pages = {e12863},
pmid = {32871045},
issn = {1365-2184},
support = {XDA16030403//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; XDA16030801//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; XDA16030303//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 2017YFA0104401//National Key Research and Development Program/ ; 2016YFA0100202//National Key Research and Development Program/ ; 2017YFA0104402//National Key Research and Development Program/ ; 31621004//National Natural Science Foundation of China/ ; QYZDB-SSW-SMC022//Key Research Projects of the Frontier Science of the Chinese Academy of Sciences/ ; },
mesh = {Animals ; Animals, Genetically Modified/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Disease Models, Animal ; Gene Editing ; Humans ; Immune System/metabolism ; Interleukin Receptor Common gamma Subunit/antagonists &amp; inhibitors/genetics/*metabolism ; Lymphopenia/pathology ; Melanoma/metabolism/pathology ; RNA Interference ; RNA, Small Interfering/metabolism ; Skin Neoplasms/metabolism/mortality/*pathology ; Survival Rate ; Swine ; Swine, Miniature ; Transplantation, Heterologous ; },
abstract = {OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development.<br><br>MATERIALS AND METHODS: IL2RG-/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis.<br><br>RESULTS: Compared to age-matched wild-type pigs, IL2RG-/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG-/Y pigs but not in wild-type littermates. The human tumours grew faster in IL2RG-/Y pigs than in nude mice.<br><br>CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.},
}
@article {pmid32870981,
year = {2020},
author = {Smith, EA and Newton, ILG},
title = {Genomic Signatures of Honey Bee Association in an Acetic Acid Symbiont.},