@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 = {},
number = {},
pages = {},
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 {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 & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2020.10.010},
pmid = {33217332},
issn = {1934-6069},
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.

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.

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.

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 {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.},
}

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

@article {pmid32178548,
year = {2020},
author = {Marchev, AS and Yordanova, ZP and Georgiev, MI},
title = {Green (cell) factories for advanced production of plant secondary metabolites.},
journal = {Critical reviews in biotechnology},
volume = {40},
number = {4},
pages = {443-458},
doi = {10.1080/07388551.2020.1731414},
pmid = {32178548},
issn = {1549-7801},
mesh = {*Bioreactors ; CRISPR-Cas Systems ; Gene Editing ; Metabolic Engineering/*methods ; Plant Cells/*metabolism ; Plants/genetics/metabolism ; *Secondary Metabolism ; },
abstract = {For centuries plants have been intensively utilized as reliable sources of food, flavoring, agrochemical and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to climate change and agriculture. Plant biotechnology offers a sustainable method for the bioproduction of plant secondary metabolites using plant in vitro systems. The unique structural features of plant-derived secondary metabolites, such as their safety profile, multi-target spectrum and "metabolite likeness," have led to the establishment of many plant-derived drugs, comprising approximately a quarter of all drugs approved by the Food and Drug Administration and/or European Medicinal Agency. However, there are still many challenges to overcome to enhance the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant secondary metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this review, we present an integrated overview of the possible avenues for enhancing the biosynthesis of high-value marketable molecules produced by plant in vitro systems. These include metabolic engineering and CRISPR/Cas9 technology for the regulation of plant metabolism through overexpression/repression of single or multiple structural genes or transcriptional factors. The use of NMR-based metabolomics for monitoring metabolite concentrations and additionally as a tool to study the dynamics of plant cell metabolism and nutritional management is discussed here. Different types of bioreactor systems, their modification and optimal process parameters for the lab- or industrial-scale production of plant secondary metabolites are specified.},
}

*Bioreactors
CRISPR-Cas Systems
Gene Editing
Metabolic Engineering/*methods
Plant Cells/*metabolism
Plants/genetics/metabolism
*Secondary Metabolism

@article {pmid32042051,
year = {2020},
author = {Chillappagari, S and Belapurkar, R and Möller, A and Molenda, N and Kracht, M and Rohrbach, S and Schmitz, ML},
title = {SIAH2-mediated and organ-specific restriction of HO-1 expression by a dual mechanism.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2268},
pmid = {32042051},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Down-Regulation ; Ferroptosis ; Fibroblasts ; Gene Knockdown Techniques ; HEK293 Cells ; Heme Oxygenase-1/genetics/*metabolism ; Humans ; Membrane Proteins/genetics/*metabolism ; Mice ; Mice, Knockout ; NF-E2-Related Factor 2/*metabolism ; Nuclear Proteins/genetics/*metabolism ; Oxygen/metabolism ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism ; Primary Cell Culture ; Proteasome Endopeptidase Complex/metabolism ; Protein Domains ; Protein Stability ; Proteolysis ; Ubiquitin-Protein Ligases/genetics/*metabolism ; Ubiquitination ; },
abstract = {The intracellular levels of the cytoprotective enzyme heme oxygenase-1 (HO-1) are tightly controlled. Here, we reveal a novel mechanism preventing the exaggerated expression of HO-1. The analysis of mice with a knock-out in the ubiquitin E3 ligase seven in absentia homolog 2 (SIAH2) showed elevated HO-1 protein levels in specific organs such as heart, kidney and skeletal muscle. Increased HO-1 protein amounts were also seen in human cells deleted for the SIAH2 gene. The higher HO-1 levels are not only due to an increased protein stability but also to elevated expression of the HO-1 encoding HMOX1 gene, which depends on the transcription factor nuclear factor E2-related factor 2 (NRF2), a known SIAH2 target. Dependent on its RING (really interesting new gene) domain, expression of SIAH2 mediates proteasome-dependent degradation of its interaction partner HO-1. Additionally SIAH2-deficient cells are also characterized by reduced expression levels of glutathione peroxidase 4 (GPX4), rendering the knock-out cells more sensitive to ferroptosis.},
}

Animals
CRISPR-Cas Systems/genetics
Down-Regulation
Ferroptosis
Fibroblasts
Gene Knockdown Techniques
HEK293 Cells
Heme Oxygenase-1/genetics/*metabolism
Humans
Membrane Proteins/genetics/*metabolism
Mice
Mice, Knockout
NF-E2-Related Factor 2/*metabolism
Nuclear Proteins/genetics/*metabolism
Oxygen/metabolism
Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism
Primary Cell Culture
Proteasome Endopeptidase Complex/metabolism
Protein Domains
Protein Stability
Proteolysis
Ubiquitin-Protein Ligases/genetics/*metabolism
Ubiquitination

@article {pmid32042045,
year = {2020},
author = {Wang, Y and Wang, M and Zheng, T and Hou, Y and Zhang, P and Tang, T and Wei, J and Du, Q},
title = {Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2269},
pmid = {32042045},
issn = {2045-2322},
mesh = {Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; Endodeoxyribonucleases/metabolism ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mutation ; Nucleotides/metabolism ; RNA, Guide/*genetics ; },
abstract = {To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a "core" sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of 'super' off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.},
}

Bacterial Proteins/metabolism
CRISPR-Associated Protein 9/metabolism
CRISPR-Associated Proteins/metabolism
CRISPR-Cas Systems/*genetics
Endodeoxyribonucleases/metabolism
Gene Editing/*methods
HEK293 Cells
Humans
Mutation
Nucleotides/metabolism
RNA, Guide/*genetics

@article {pmid32029872,
year = {2020},
author = {Kitamoto, K and Taketani, Y and Fujii, W and Inamochi, A and Toyono, T and Miyai, T and Yamagami, S and Kuroda, M and Usui, T and Ouchi, Y},
title = {Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2000},
pmid = {32029872},
issn = {2045-2322},
mesh = {Amino Acid Substitution ; Animals ; Arginine/genetics ; CRISPR-Cas Systems ; Corneal Dystrophies, Hereditary/*genetics/pathology ; Corneal Stroma/pathology/ultrastructure ; Cysteine/genetics ; *Disease Models, Animal ; Extracellular Matrix Proteins/*genetics ; Female ; Heterozygote ; Homozygote ; Humans ; Male ; Mice ; Mice, Transgenic ; Microscopy, Electron, Transmission ; Mutation ; Recombinational DNA Repair ; Transforming Growth Factor beta/*genetics ; },
abstract = {Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.},
}

Amino Acid Substitution
Animals
Arginine/genetics
CRISPR-Cas Systems
Corneal Dystrophies, Hereditary/*genetics/pathology
Corneal Stroma/pathology/ultrastructure
Cysteine/genetics
*Disease Models, Animal
Extracellular Matrix Proteins/*genetics
Female
Heterozygote
Homozygote
Humans
Male
Mice
Mice, Transgenic
Microscopy, Electron, Transmission
Mutation
Recombinational DNA Repair
Transforming Growth Factor beta/*genetics

@article {pmid31761839,
year = {2020},
author = {Yamashita, S and Kogasaka, Y and Hiradate, Y and Tanemura, K and Sendai, Y},
title = {Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation.},
journal = {The Journal of reproduction and development},
volume = {66},
number = {1},
pages = {41-48},
pmid = {31761839},
issn = {1348-4400},
mesh = {Animals ; Blastocyst/*metabolism ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electroporation ; Embryonic Development/*physiology ; Exodeoxyribonucleases/*genetics ; Gene Editing/*methods ; *Mosaicism ; Mutation ; Phosphoproteins/*genetics ; Swine ; Zygote/*metabolism ; },
abstract = {Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.},
}

Animals
Blastocyst/*metabolism
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Electroporation
Embryonic Development/*physiology
Exodeoxyribonucleases/*genetics
Gene Editing/*methods
*Mosaicism
Mutation
Phosphoproteins/*genetics
Swine
Zygote/*metabolism

@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},
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 {pmid32761584,
year = {2020},
author = {Gao, W and Liu, H and Su, G and Xu, Y and Wang, Y and Cui, L and Huang, R and Yang, H and Gao, M and Xi, S and Shen, G},
title = {[Development of a cell-based diagnostic system for vitamin K-dependent coagulation factor deficiency 1].},
journal = {Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics},
volume = {37},
number = {8},
pages = {811-814},
doi = {10.3760/cma.j.issn.1003-9406.2020.08.002},
pmid = {32761584},
issn = {1003-9406},
mesh = {Base Sequence ; Blood Coagulation Disorders, Inherited/diagnosis/*genetics ; CRISPR-Cas Systems ; Carbon-Carbon Ligases/*genetics ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; *Vitamin K 1 ; },
abstract = {OBJECTIVE: To develop a cell-based system for the diagnosis of vitamin K-dependent coagulation factor deficiency 1 (VKCFD1).

METHODS: In HEK293 cells stably expressing the reporter gene FIX-Gla-PC, the gamma-glutamyl carboxylase (GGCX) gene was knocked out by using CRISPR/Cas9 technology. Enzyme-linked immunosorbent assay (ELISA), DNA sequencing and Western blotting were used to identify the GGCX gene knockout cells. A quickchange point variant method was used to construct the GGCX variant. ELISA was used to assess the influence of GGCX variant on the activity of reporter gene.

RESULTS: Two monoclonal cell lines with no reporter activity by ELISA was identified. Edition and knockout of the GGCX gene was confirmed by DNA sequencing and Western blotting. The activity of the reporter gene was recovered by transfection of the wild-type GGCX gene. Thereby two monoclonal cells with GGCX knockout were obtained. By comparing the wild-type and pathogenic GGCX variants, the reporter activity was decreased in the pathogenic variants significantly.

CONCLUSION: A cell-based system for the detection of GGCX activity was successfully developed, which can be used for the diagnosis of VKCFD1 caused by GGCX variants.},
}

Base Sequence
Blood Coagulation Disorders, Inherited/diagnosis/*genetics
CRISPR-Cas Systems
Carbon-Carbon Ligases/*genetics
Gene Knockout Techniques
HEK293 Cells
Humans
*Vitamin K 1

@article {pmid32093670,
year = {2020},
author = {Michno, JM and Virdi, K and Stec, AO and Liu, J and Wang, X and Xiong, Y and Stupar, RM},
title = {Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines.},
journal = {BMC biotechnology},
volume = {20},
number = {1},
pages = {10},
pmid = {32093670},
issn = {1472-6750},
support = {2015-33522-24096//National Institute of Food and Agriculture/International ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing ; High-Throughput Nucleotide Sequencing ; *Mutation ; Plants, Genetically Modified/growth & development ; Quantitative Trait, Heritable ; Sequence Analysis, DNA ; Soybeans/*genetics/growth & development ; Transgenes ; },
abstract = {BACKGROUND: As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results.

RESULTS: In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission.

CONCLUSIONS: Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.},
}

*CRISPR-Cas Systems
Gene Editing
High-Throughput Nucleotide Sequencing
*Mutation
Plants, Genetically Modified/growth & development
Quantitative Trait, Heritable
Sequence Analysis, DNA
Soybeans/*genetics/growth & development
Transgenes

@article {pmid32046096,
year = {2020},
author = {Wu, N and Lu, Q and Wang, P and Zhang, Q and Zhang, J and Qu, J and Wang, N},
title = {Construction and Analysis of GmFAD2-1A and GmFAD2-2A Soybean Fatty Acid Desaturase Mutants Based on CRISPR/Cas9 Technology.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {32046096},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/*genetics/metabolism ; Fatty Acid Desaturases/chemistry/*genetics/metabolism ; Gene Editing/methods ; *Mutation ; Oleic Acid/biosynthesis ; Plant Breeding/methods ; Plant Proteins/chemistry/*genetics/metabolism ; Seeds/genetics/metabolism ; Soybeans/*genetics/metabolism ; },
abstract = {The soybean fatty acid desaturase family is composed of seven genes, but the function of each gene has not been reported. Bioinformatics was used to analyse the structure of genes in this family, as well as the correlation between Δ12-fatty acid desaturase II (FAD2) expression and oleic acid content on different days after flowering of soybean. In the present study, CRISPR/Cas9 technology was used to construct single and double mutant knockout vectors of functional genes in the FAD2 family. Analysis of the molecular biology and expression patterns of genes in the FAD2 family, namely, GmFAD2-1A (Glyma.10G278000) and GmFAD2-2A (Glyma.19G147300), showed that they had little homology with other soybean FAD2 genes, and that their function was slightly changed. Sequencing of the target showed that the editing efficiency of the GmFAD2-1A and GmFAD2-2A genes was 95% and 55.56%, respectively, and that the double mutant editing efficiency was 66.67%. The mutations were divided into two main types, as follows: base deletion and insertion. A near-infrared grain analyser determined the following results: In the T2 generation, the oleic acid content increased from 17.10% to 73.50%; the linoleic acid content decreased from 62.91% to 12.23%; the protein content increased from 37.69% to 41.16%; in the T3 generation, the oleic acid content increased from 19.15% to 72.02%; the linoleic acid content decreased from 56.58% to 17.27%. In addition, the protein content increased from 37.52% to 40.58% compared to that of the JN38 control variety.},
}

*CRISPR-Cas Systems
Crops, Agricultural/*genetics/metabolism
Fatty Acid Desaturases/chemistry/*genetics/metabolism
Gene Editing/methods
*Mutation
Oleic Acid/biosynthesis
Plant Breeding/methods
Plant Proteins/chemistry/*genetics/metabolism
Seeds/genetics/metabolism
Soybeans/*genetics/metabolism

@article {pmid32029861,
year = {2020},
author = {Sevestre, F and Facon, M and Wattebled, F and Szydlowski, N},
title = {Facilitating gene editing in potato: a Single-Nucleotide Polymorphism (SNP) map of the Solanum tuberosum L. cv. Desiree genome.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2045},
pmid = {32029861},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Gene Knockout Techniques ; Genome, Plant/*genetics ; High-Throughput Nucleotide Sequencing ; Molecular Sequence Annotation ; Mutagenesis ; Plant Proteins/genetics ; Plants, Genetically Modified ; Polymorphism, Single Nucleotide ; RNA, Guide/genetics ; *Reverse Genetics ; Solanum tuberosum/*genetics ; Starch Synthase/genetics ; },
abstract = {Genome editing is a powerful tool for plant functional genomics allowing for multiallelic targeted mutagenesis. The recent development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) systems for gene editing in plants allows for simple, cost-effective introduction of site-specific double-stranded DNA breaks. The nuclear genomes of a homozygous doubled-monoploid potato clone (DM) and a heterozygous diploid clone (RH) have been sequenced in 2011. However, common potato cultivars display a highly heterozygous autotetraploid genome thus complicating target design for tetra-allelic gene editing. Here, we report on the SNP physical map of the widely used Solanum tuberosum L. cv. Desiree and on the position of the diverse indels providing an essential tool for target design in genome editing approaches. We used this tool for designing a specific gRNA and successfully knocking-out a newly discovered starch synthase gene (SS6) in potato. Resequencing data are publicly available at the Sequence Read Archive of the NCBI (accession number: PRJNA507597) and will represent a valuable resource for functional genomic studies of various metabolic pathways, cell and plant physiology as well as high-throughput reverse genetics in potato.},
}

CRISPR-Cas Systems/genetics
Gene Editing/*methods
Gene Knockout Techniques
Genome, Plant/*genetics
High-Throughput Nucleotide Sequencing
Molecular Sequence Annotation
Mutagenesis
Plant Proteins/genetics
Plants, Genetically Modified
Polymorphism, Single Nucleotide
RNA, Guide/genetics
*Reverse Genetics
Solanum tuberosum/*genetics
Starch Synthase/genetics

@article {pmid32029769,
year = {2020},
author = {Wessel, GM and Kiyomoto, M and Shen, TL and Yajima, M},
title = {Genetic manipulation of the pigment pathway in a sea urchin reveals distinct lineage commitment prior to metamorphosis in the bilateral to radial body plan transition.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1973},
pmid = {32029769},
issn = {2045-2322},
support = {R01 GM125071/GM/NIGMS NIH HHS/United States ; R01 GM126043/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Biosynthetic Pathways/genetics ; Body Patterning/*physiology ; CRISPR-Cas Systems/genetics ; Cell Lineage ; Embryo, Nonmammalian ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; *Metamorphosis, Biological ; Oxygenases/genetics/metabolism ; Pigmentation/*physiology ; Pigments, Biological/*biosynthesis ; Polyketide Synthases/genetics/metabolism ; Polyketides/metabolism ; Sea Urchins/*growth & development ; Transcription Factors/genetics/metabolism ; },
abstract = {Echinoderms display a vast array of pigmentation and patterning in larval and adult life stages. This coloration is thought to be important for immune defense and camouflage. However, neither the cellular nor molecular mechanism that regulates this complex coloration in the adult is known. Here we knocked out three different genes thought to be involved in the pigmentation pathway(s) of larvae and grew the embryos to adulthood. The genes tested were polyketide synthase (PKS), Flavin-dependent monooxygenase family 3 (FMO3) and glial cells missing (GCM). We found that disabling of the PKS gene at fertilization resulted in albinism throughout all life stages and throughout all cells and tissues of this animal, including the immune cells of the coelomocytes. We also learned that FMO3 is an essential modifier of the polyketide. FMO3 activity is essential for larval pigmentation, but in juveniles and adults, loss of FMO3 activity resulted in the animal becoming pastel purple. Linking the LC-MS analysis of this modified pigment to a naturally purple animal suggested a conserved echinochrome profile yielding a pastel purple. We interpret this result as FMO3 modifies the parent polyketide to contribute to the normal brown/green color of the animal, and that in its absence, other biochemical modifications are revealed, perhaps by other members of the large FMO family in this animal. The FMO modularity revealed here may be important in the evolutionary changes between species and for different immune challenges. We also learned that glial cells missing (GCM), a key transcription factor of the endomesoderm gene regulatory network of embryos in the sea urchin, is required for pigmentation throughout the life stages of this sea urchin, but surprisingly, is not essential for larval development, metamorphosis, or maintenance of adulthood. Mosaic knockout of either PKS or GCM revealed spatial lineage commitment in the transition from bilaterality of the larva to a pentaradial body plan of the adult. The cellular lineages identified by pigment presence or absence (wild-type or knock-out lineages, respectively) followed a strict oral/aboral profile. No circumferential segments were seen and instead we observed 10-fold symmetry in the segments of pigment expression. This suggests that the adult lineage commitments in the five outgrowths of the hydropore in the larva are early, complete, fixed, and each bilaterally symmetric. Overall, these results suggest that pigmentation of this animal is genetically determined and dependent on a population of pigment stem cells that are set-aside in a sub-region of each outgrowth of the pentaradial adult rudiment prior to metamorphosis. This study reveals the complex chemistry of pigment applicable to many organisms, and further, provides an insight into the key transitions from bilateral to pentaradial body plans unique to echinoderms.},
}

Animals
Biosynthetic Pathways/genetics
Body Patterning/*physiology
CRISPR-Cas Systems/genetics
Cell Lineage
Embryo, Nonmammalian
Gene Expression Regulation, Developmental
Gene Knockout Techniques
*Metamorphosis, Biological
Oxygenases/genetics/metabolism
Pigmentation/*physiology
Pigments, Biological/*biosynthesis
Polyketide Synthases/genetics/metabolism
Polyketides/metabolism
Sea Urchins/*growth & development
Transcription Factors/genetics/metabolism

@article {pmid31974430,
year = {2020},
author = {Sakurai, T and Kamiyoshi, A and Kawate, H and Watanabe, S and Sato, M and Shindo, T},
title = {Production of genetically engineered mice with higher efficiency, lower mosaicism, and multiplexing capability using maternally expressed Cas9.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1091},
pmid = {31974430},
issn = {2045-2322},
support = {16K15233//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; 19K07477//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; },
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Gene Targeting ; Male ; Mice/*genetics ; Mice, Inbred ICR ; Mice, Transgenic ; *Mosaicism ; },
abstract = {The CRISPR/Cas9 system is widely used to generate gene-edited animals. Here, we developed an efficient system for generating genetically modified mice using maternal Cas9 from Cas9 transgenic mice. Using this system, we achieved lower mosaicism and higher rates of knock-in success, gene-editing, and birth compared to the similar parameters obtained using exogenously administered Cas9 (mRNA/protein) system. Furthermore, we successfully induced simultaneous mutations at multiple loci (a maximum of nine). Our novel gene-editing system based on maternal Cas9 could potentially facilitate the generation of mice with single and multiple gene modifications.},
}

Animals
Animals, Genetically Modified/genetics
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems
Female
Gene Editing/*methods
Gene Targeting
Male
Mice/*genetics
Mice, Inbred ICR
Mice, Transgenic
*Mosaicism

@article {pmid31460749,
year = {2019},
author = {Wang, B and Wang, R and Wang, D and Wu, J and Li, J and Wang, J and Liu, H and Wang, Y},
title = {Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {91},
number = {19},
pages = {12156-12161},
doi = {10.1021/acs.analchem.9b01526},
pmid = {31460749},
issn = {1520-6882},
mesh = {Bacterial Proteins/chemistry/genetics ; Buffers ; CRISPR-Associated Proteins/chemistry/genetics ; *CRISPR-Cas Systems ; DNA/*analysis ; Electrophoresis, Agar Gel ; Endodeoxyribonucleases/chemistry/genetics ; Mycoplasma/genetics ; Nucleic Acid Amplification Techniques/*methods ; },
abstract = {A rapid and sensitive method is crucial for nucleic acid detection. Recently, RNA-guided CRISPR/Cas12a nuclease-based methods present great promise for nucleic acid detection. In the present methods, however, DNA amplification and subsequent Cas12a cleavage is separated and the whole process takes as long as 2 h. Most importantly, the uncapping operation increases the risk of aerosol contamination. In this study, we propose a CRISPR/Cas12a-based method named "Cas12aVDet" for rapid nucleic acid detection. By integrating recombinase polymerase amplification (RPA) with Cas12a cleavage in a single reaction system, the detection can be accomplished in 30 min and uncapping contamination can be avoided. The detection signal can be observed by the naked eye under blue light. This method could detect DNA at single molecule level and demonstrated 100% accuracy for mycoplasma contamination detection, presenting great potential for a variety of nucleic acid detection applications.},
}

Bacterial Proteins/chemistry/genetics
Buffers
CRISPR-Associated Proteins/chemistry/genetics
*CRISPR-Cas Systems
DNA/*analysis
Electrophoresis, Agar Gel
Endodeoxyribonucleases/chemistry/genetics
Mycoplasma/genetics
Nucleic Acid Amplification Techniques/*methods

@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 = {},
doi = {10.3390/microorganisms8111775},
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 {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.},
}

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

@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 ; },
}

CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Deoxyribonuclease I
*Gene Editing
RNA

@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.},
}

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

@article {pmid32821003,
year = {2020},
author = {Assimos, DG},
title = {Re: CRISPR/Cas9-Mediated Metabolic Pathway Reprogramming in a Novel Humanized Rat Model Ameliorates Primary Hyperoxaluria Type 1.},
journal = {The Journal of urology},
volume = {204},
number = {5},
pages = {1092},
doi = {10.1097/JU.0000000000001258.04},
pmid = {32821003},
issn = {1527-3792},
mesh = {Animals ; *CRISPR-Cas Systems ; *Hyperoxaluria, Primary/genetics ; Metabolic Networks and Pathways ; Rats ; },
}

Animals
*CRISPR-Cas Systems
*Hyperoxaluria, Primary/genetics
Metabolic Networks and Pathways
Rats

@article {pmid32575125,
year = {2020},
author = {Zeng, Z and Han, N and Liu, C and Buerte, B and Zhou, C and Chen, J and Wang, M and Zhang, Y and Tang, Y and Zhu, M and Wang, J and Yang, Y and Bian, H},
title = {Functional dissection of HGGT and HPT in barley vitamin E biosynthesis via CRISPR/Cas9-enabled genome editing.},
journal = {Annals of botany},
volume = {126},
number = {5},
pages = {929-942},
pmid = {32575125},
issn = {1095-8290},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Hordeum/genetics ; Humans ; Tocopherols ; *Tocotrienols ; Vitamin E ; },
abstract = {BACKGROUND AND AIMS: Vitamin E (tocochromanol) is a lipid-soluble antioxidant and an essential nutrient for human health. Among cereal crops, barley (Hordeum vulgare) contains a high level of vitamin E, which includes both tocopherols and tocotrienols. Although the vitamin E biosynthetic pathway has been characterized in dicots, such as Arabidopsis, which only accumulate tocopherols, knowledge regarding vitamin E biosynthesis in monocots is limited because of the lack of functional mutants. This study aimed to obtain gene knockout mutants to elucidate the genetic control of vitamin E composition in barley.

METHODS: Targeted knockout mutations of HvHPT and HvHGGT in barley were created with CRISPR/Cas9-enabled genome editing. High-performance liquid chromatography (HPLC) was performed to analyse the content of tocochromanol isomers in transgene-free homozygous Hvhpt and Hvhggt mutants.

KEY RESULTS: Mutagenesis efficiency among T0 regenerated plantlets was 50-65 % as a result of two simultaneously expressed guide RNAs targeting each gene; most of the mutations were stably inherited by the next generation. The transgene-free homozygous mutants of Hvhpt and Hvhggt exhibited decreased grain size and weight, and the HvHGGT mutation led to a shrunken phenotype and significantly lower total starch content in grains. HPLC analysis revealed that targeted mutation of HvHPT significantly reduced the content of both tocopherols and tocotrienols, whereas mutations in HvHGGT completely blocked tocotrienol biosynthesis in barley grains. Transient overexpression of an HvHPT homologue in tobacco leaves significantly increased the production of γ- and δ-tocopherols, which may partly explain why targeted mutation of HvHPT in barley grains did not eliminate tocopherol production.

CONCLUSIONS: Our results functionally validated that HvHGGT is the only committed gene for the production of tocotrienols, whereas HvHPT is partly responsible for tocopherol biosynthesis in barley.},
}

CRISPR-Cas Systems/genetics
Gene Editing
*Hordeum/genetics
Humans
Tocopherols
*Tocotrienols
Vitamin E

@article {pmid32541946,
year = {2020},
author = {Costafreda, MI and Abbasi, A and Lu, H and Kaplan, G},
title = {Exosome mimicry by a HAVCR1-NPC1 pathway of endosomal fusion mediates hepatitis A virus infection.},
journal = {Nature microbiology},
volume = {5},
number = {9},
pages = {1096-1106},
doi = {10.1038/s41564-020-0740-y},
pmid = {32541946},
issn = {2058-5276},
support = {FD999999/ImFDA/Intramural FDA HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Carrier Proteins/metabolism ; Cell Line ; Ebolavirus ; Endocytosis ; Endosomes/*metabolism/virology ; Exosomes/*metabolism/virology ; Gene Knockout Techniques ; HEK293 Cells ; Hepatitis A/immunology/*metabolism/virology ; Hepatitis A Virus Cellular Receptor 1/genetics/*metabolism ; Hepatitis A virus/*metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Membrane Glycoproteins ; Transcriptome ; Viral Fusion Proteins/metabolism ; Virion/metabolism ; Virus Internalization ; },
abstract = {Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis, binding and uptake of exosomes are well characterized4,5, delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology, we show that these two lipid receptors, which interact in the late endosome9, are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway, which Ebola virus exploits to infect cells9, mediates HAV infection by exo-HAV, which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen, but not the endosomal uncoating of HAV particles contained in the exosomes, is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV, infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses, and call for a reassessment of the role of envelope glycoproteins in infection.},
}

CRISPR-Cas Systems
Carrier Proteins/metabolism
Cell Line
Ebolavirus
Endocytosis
Endosomes/*metabolism/virology
Exosomes/*metabolism/virology
Gene Knockout Techniques
HEK293 Cells
Hepatitis A/immunology/*metabolism/virology
Hepatitis A Virus Cellular Receptor 1/genetics/*metabolism
Hepatitis A virus/*metabolism
Humans
Intracellular Signaling Peptides and Proteins/genetics/*metabolism
Membrane Glycoproteins
Transcriptome
Viral Fusion Proteins/metabolism
Virion/metabolism
Virus Internalization

@article {pmid32497313,
year = {2020},
author = {Nateghi Rostami, M},
title = {CRISPR/Cas9 gene drive technology to control transmission of vector-borne parasitic infections.},
journal = {Parasite immunology},
volume = {42},
number = {9},
pages = {e12762},
doi = {10.1111/pim.12762},
pmid = {32497313},
issn = {1365-3024},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Drive Technology ; Humans ; *Insect Vectors ; Malaria/*prevention & control/transmission ; },
abstract = {Gene drive is the process of copying of an endonuclease-containing cassette that leads to increased frequency of inheritance of the desired traits in a targeted population. CRISPR/Cas9 technology is advancing genetic manipulation of insects in the field of gene drive experiments. The CRISPR/Cas9 drive could be engineered for genetic manipulation of parasites and/or vectors for disease control. A number of promising CRISPR/Cas9-based gene drive strategies that interfere with parasite development or impairs the reproductive capability of the insect vector have been proposed in the laboratory for blocking transmission of malaria and leishmaniasis. Still several technical and ethical challenges remain to be addressed, and none appear insuperable in this field.},
}

Animals
*CRISPR-Cas Systems
*Gene Drive Technology
Humans
*Insect Vectors
Malaria/*prevention & control/transmission

@article {pmid32424339,
year = {2020},
author = {Guo, R and Zhang, Y and Teng, M and Jiang, C and Schineller, M and Zhao, B and Doench, JG and O'Reilly, RJ and Cesarman, E and Giulino-Roth, L and Gewurz, BE},
title = {DNA methylation enzymes and PRC1 restrict B-cell Epstein-Barr virus oncoprotein expression.},
journal = {Nature microbiology},
volume = {5},
number = {8},
pages = {1051-1063},
doi = {10.1038/s41564-020-0724-y},
pmid = {32424339},
issn = {2058-5276},
support = {R35 CA047006/CA/NCI NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; R01 CA228700/CA/NCI NIH HHS/United States ; R01 CA047006/CA/NCI NIH HHS/United States ; R01 AI137337/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, Viral ; B-Lymphocytes/*virology ; Burkitt Lymphoma ; CCAAT-Enhancer-Binding Proteins ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism/*pharmacology ; DNA (Cytosine-5-)-Methyltransferase 1/metabolism ; DNA (Cytosine-5-)-Methyltransferases ; DNA Methylation/*physiology ; Epstein-Barr Virus Nuclear Antigens ; Gene Expression Regulation, Viral/*drug effects ; Genes, Viral ; Genome, Viral ; Herpesvirus 4, Human/*drug effects/*genetics/metabolism ; Humans ; Oncogene Proteins/genetics/*metabolism ; Ubiquitin-Protein Ligases ; },
abstract = {To accomplish the remarkable task of lifelong infection, the Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programmes to navigate the B-cell compartment and evade immune responses. The transforming programme, consisting of highly immunogenic EBV nuclear antigen (EBNA) and latent membrane proteins (LMPs), is expressed in newly infected B lymphocytes and in post-transplant lymphomas. On memory cell differentiation and in most EBV-associated Burkitt's lymphomas, all but one viral antigen are repressed for immunoevasion. To gain insights into the epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR-Cas9 screen was performed in EBV and Burkitt's lymphoma cells. Here, we show that the ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and its DNA methyltransferase partner DNA methyltransferase I (DNMT1) are critical for the restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency programme switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal centre B cells, the Burkitt's lymphoma cell of origin, providing a molecular link between B-cell state and the EBV latency programme. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.},
}

Antigens, Viral
B-Lymphocytes/*virology
Burkitt Lymphoma
CCAAT-Enhancer-Binding Proteins
CRISPR-Cas Systems
Cell Cycle Proteins/genetics/*metabolism/*pharmacology
DNA (Cytosine-5-)-Methyltransferase 1/metabolism
DNA (Cytosine-5-)-Methyltransferases
DNA Methylation/*physiology
Epstein-Barr Virus Nuclear Antigens
Gene Expression Regulation, Viral/*drug effects
Genes, Viral
Genome, Viral
Herpesvirus 4, Human/*drug effects/*genetics/metabolism
Humans
Oncogene Proteins/genetics/*metabolism
Ubiquitin-Protein Ligases

@article {pmid32033083,
year = {2020},
author = {Veillet, F and Perrot, L and Guyon-Debast, A and Kermarrec, MP and Chauvin, L and Chauvin, JE and Gallois, JL and Mazier, M and Nogué, F},
title = {Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {32033083},
issn = {1422-0067},
support = {ANR-11-BTBR-0001_GENIUS//Agence Nationale de la Recherche/ ; ANR-10-LABX-0040-SPS//Agence Nationale de la Recherche/ ; },
mesh = {Amino Acid Substitution/genetics ; Bryopsida/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Gene Editing/*methods ; Lycopersicon esculentum/*genetics ; Plants, Genetically Modified/genetics ; Solanum tuberosum/*genetics ; Streptococcus pyogenes/enzymology ; },
abstract = {Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrellapatens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.},
}

Amino Acid Substitution/genetics
Bryopsida/*genetics
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/genetics
Crops, Agricultural/genetics
Gene Editing/*methods
Lycopersicon esculentum/*genetics
Plants, Genetically Modified/genetics
Solanum tuberosum/*genetics
Streptococcus pyogenes/enzymology

@article {pmid32028669,
year = {2020},
author = {Zhao, W and Wang, Y and Liang, FS},
title = {Chemical and Light Inducible Epigenome Editing.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
pmid = {32028669},
issn = {1422-0067},
support = {R21HG010673/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Methylation ; *Epigenesis, Genetic ; *Epigenome ; Gene Editing/*methods ; Humans ; *Light ; Small Molecule Libraries/*pharmacology ; },
abstract = {The epigenome defines the unique gene expression patterns and resulting cellular behaviors in different cell types. Epigenome dysregulation has been directly linked to various human diseases. Epigenome editing enabling genome locus-specific targeting of epigenome modifiers to directly alter specific local epigenome modifications offers a revolutionary tool for mechanistic studies in epigenome regulation as well as the development of novel epigenome therapies. Inducible and reversible epigenome editing provides unique temporal control critical for understanding the dynamics and kinetics of epigenome regulation. This review summarizes the progress in the development of spatiotemporal-specific tools using small molecules or light as inducers to achieve the conditional control of epigenome editing and their applications in epigenetic research.},
}

Animals
*CRISPR-Cas Systems
*DNA Methylation
*Epigenesis, Genetic
*Epigenome
Gene Editing/*methods
Humans
*Light
Small Molecule Libraries/*pharmacology

@article {pmid32020014,
year = {2020},
author = {Xu, D and Cai, Y and Tang, L and Han, X and Gao, F and Cao, H and Qi, F and Kapranov, P},
title = {A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1794},
pmid = {32020014},
issn = {2045-2322},
support = {31671382//National Natural Science Foundation of China (National Science Foundation of China)/International ; },
mesh = {Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/methods ; Humans ; RNA, Long Noncoding/genetics/*metabolism ; },
abstract = {Long non-coding (lnc) RNAs represent a fascinating class of transcripts that remains highly controversial mainly due to ambiguity surrounding overall biological relevance of these RNAs. Multitude of reverse genetics studies showing functionality of lncRNAs are unfortunately based on assays that are either plagued by non-specific effects and/or cannot unambiguously assign observed phenotypes to the transcript per se. Here, we show application of the novel CRISPR/Cas13 RNA knockdown system that has superior specificity compared to other transcript-targeting knockdown methods like RNAi. We applied this method to a novel widespread subclass of nuclear lncRNAs - very long intergenic non-coding (vlinc) RNAs - in a high-throughput phenotypic assay based on survival challenge in response to anticancer drug treatments. We used multiple layers of controls including mismatch control for each targeting gRNA to ensure uncovering true phenotype-transcript relationships. We found evidence supporting importance for cellular survival for up to 60% of the tested protein-coding mRNAs and, importantly, 64% of vlincRNAs. Overall, this study demonstrates utility of CRISPR/Cas13 as a highly sensitive and specific tool for reverse genetics study of both protein-coding genes and lncRNAs. Furthermore, importantly, this approach provides evidence supporting biological significance of the latter transcripts in anticancer drug response.},
}

Antineoplastic Agents/*pharmacology
CRISPR-Cas Systems
Cell Line, Tumor
Cell Survival/drug effects
*Clustered Regularly Interspaced Short Palindromic Repeats
Computational Biology/methods
Humans
RNA, Long Noncoding/genetics/*metabolism

@article {pmid32005926,
year = {2020},
author = {de Almeida Monteiro Melo Ferraz, M and Nagashima, JB and Venzac, B and Le Gac, S and Songsasen, N},
title = {A dog oviduct-on-a-chip model of serous tubal intraepithelial carcinoma.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1575},
pmid = {32005926},
issn = {2045-2322},
support = {F32 HD090854/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Carcinoma in Situ/metabolism/*veterinary ; Dog Diseases/*metabolism ; Dogs ; Female ; Fluorescent Antibody Technique ; Gene Editing ; Lab-On-A-Chip Devices/*veterinary ; Ovarian Neoplasms/metabolism/*veterinary ; Oviducts/*metabolism ; Polymerase Chain Reaction ; },
abstract = {Ovarian cancer is the fifth cause of cancer-related mortality in women, with an expected 5-year survival rate of only 47%. High-grade serous carcinoma (HGSC), an epithelial cancer phenotype, is the most common malignant ovarian cancer. It is known that the precursors of HGSC originate from secretory epithelial cells within the Fallopian tube, which first develops as serous tubal intraepithelial carcinoma (STIC). Here, we used gene editing by CRISPR-Cas9 to knock out the oncogene p53 in dog oviductal epithelia cultured in a dynamic microfluidic chip to create an in vitro model that recapitulated human STIC. Similar to human STIC, the gene-edited oviduct-on-a-chip, exhibited loss of cell polarization and had reduced ciliation, increased cell atypia and proliferation, with multilayered epithelium, increased Ki67, PAX8 and Myc and decreased PTEN and RB1 mRNA expression. This study provides a biomimetic in vitro model to study STIC progression and to identify potential biomarkers for early detection of HGSC.},
}

Animals
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Carcinoma in Situ/metabolism/*veterinary
Dog Diseases/*metabolism
Dogs
Female
Fluorescent Antibody Technique
Gene Editing
Lab-On-A-Chip Devices/*veterinary
Ovarian Neoplasms/metabolism/*veterinary
Oviducts/*metabolism
Polymerase Chain Reaction

@article {pmid32001790,
year = {2020},
author = {Oladimeji, PO and Bakke, J and Wright, WC and Chen, T},
title = {KANSL2 and MBNL3 are regulators of pancreatic ductal adenocarcinoma invasion.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {1485},
pmid = {32001790},
issn = {2045-2322},
support = {R35 GM118041/GM/NIGMS NIH HHS/United States ; P30-CA21765//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/International ; },
mesh = {CRISPR-Cas Systems ; Carcinoma, Pancreatic Ductal/*genetics/pathology ; Cell Line, Tumor ; Cell Movement/genetics ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Histone Acetyltransferases/antagonists & inhibitors/*genetics ; Humans ; Neoplasm Invasiveness/genetics/pathology ; Pancreatic Neoplasms/*genetics/pathology ; RNA, Small Interfering/genetics ; RNA-Binding Proteins/antagonists & inhibitors/*genetics ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer. One major reason for this is that PDAC quickly metastasizes to other organs, thereby making its treatment difficult. The molecular machinery driving PDAC metastasis is still poorly understood. In this study, we applied an unbiased approach using CRISPR screening to identify genes that strongly regulate invasion (based on an in vitro assessment of their metastatic potential) in PANC-1, a PDAC cell line. Through CRISPR screening, we identified MBNL3 and KANSL2 as strong regulators of invasion in PANC-1 cells. We further validated MBNL3 and KANSL2 as regulators of PANC-1 cell invasion by using the doxycycline-inducible shRNA system. We also showed that MBNL3 and KANSL2 do not affect cell proliferation. Through our efforts, we have established a process to identify genes that regulate cell invasion and can be further investigated as potential targets for therapeutic intervention.},
}

CRISPR-Cas Systems
Carcinoma, Pancreatic Ductal/*genetics/pathology
Cell Line, Tumor
Cell Movement/genetics
Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Histone Acetyltransferases/antagonists & inhibitors/*genetics
Humans
Neoplasm Invasiveness/genetics/pathology
Pancreatic Neoplasms/*genetics/pathology
RNA, Small Interfering/genetics
RNA-Binding Proteins/antagonists & inhibitors/*genetics

@article {pmid31941961,
year = {2020},
author = {Straume, AH and Kjærner-Semb, E and Ove Skaftnesmo, K and Güralp, H and Kleppe, L and Wargelius, A and Edvardsen, RB},
title = {Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {409},
pmid = {31941961},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; Embryo, Mammalian/cytology/metabolism ; Fish Proteins/antagonists & inhibitors/genetics/*metabolism ; Gene Editing ; *INDEL Mutation ; Membrane Transport Proteins/chemistry/genetics/*metabolism ; *Recombinational DNA Repair ; Salmo salar/embryology/*genetics ; },
abstract = {Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5'-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general.},
}

Animals
*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded
Embryo, Mammalian/cytology/metabolism
Fish Proteins/antagonists & inhibitors/genetics/*metabolism
Gene Editing
*INDEL Mutation
Membrane Transport Proteins/chemistry/genetics/*metabolism
*Recombinational DNA Repair
Salmo salar/embryology/*genetics

@article {pmid31882627,
year = {2019},
author = {Ferrara, M and Haidukowski, M and Logrieco, AF and Leslie, JF and Mulè, G},
title = {A CRISPR-Cas9 System for Genome Editing of Fusarium proliferatum.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {19836},
pmid = {31882627},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Fumonisins/*metabolism ; Fungal Proteins/genetics/metabolism ; Fusarium/*genetics/metabolism ; Gene Deletion ; Gene Editing/*methods ; Mutation ; Polyketide Synthases/genetics/metabolism ; Reproducibility of Results ; },
abstract = {Fusarium proliferatum causes diverse diseases of many economically important plants. The fungus produces several mycotoxins of which the fumonisins are the most toxic. Currently, deletion of key genes for mycotoxin biosynthesis is a laborious and time-consuming procedure. We developed a novel CRISPR/Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins into protoplasts of F. proliferatum. Our CRISPR-Cas9 system couples a site-specific double-strand DNA break mediated by two Cas9 ribonucleoproteins with microhomology recombination requiring only 50-bp regions flanking the target gene. This system reduces the risk of off-target mutations and minimizes the risk of altering any gene adjacent to the target region. We used this tool to delete a polyketide synthase gene (FUM1) required for fumonisin biosynthesis. The mutants generated are no longer able to produce fumonisins, confirming the key role of FUM1 in fumonisin biosynthesis. Our CRISPR-Cas9 system is an important new tool for genetic studies of Fusarium.},
}

*CRISPR-Cas Systems
Fumonisins/*metabolism
Fungal Proteins/genetics/metabolism
Fusarium/*genetics/metabolism
Gene Deletion
Gene Editing/*methods
Mutation
Polyketide Synthases/genetics/metabolism
Reproducibility of Results

@article {pmid31566854,
year = {2019},
author = {Lyu, Y and He, S and Li, J and Jiang, Y and Sun, H and Miao, Y and Pu, K},
title = {A Photolabile Semiconducting Polymer Nanotransducer for Near-Infrared Regulation of CRISPR/Cas9 Gene Editing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {58},
number = {50},
pages = {18197-18201},
doi = {10.1002/anie.201909264},
pmid = {31566854},
issn = {1521-3773},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Fluorescence ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Vectors ; HeLa Cells ; Humans ; Mice, Nude ; Nanostructures/*chemistry ; Photochemical Processes ; Plasmids/genetics ; Polyethylene Glycols/chemistry ; Polyethyleneimine/chemistry ; Polymers/*chemistry ; Proof of Concept Study ; Singlet Oxygen ; },
abstract = {Noninvasive regulation of CRISPR/Cas9 gene editing is conducive to understanding of gene function and development of gene therapy; however, it remains challenging. Herein, a photolabile semiconducting polymer nanotransducer (pSPN) is synthesized to act as the gene vector to deliver CRISPR/Cas9 plasmids into cells and also as the photoregulator to remotely activate gene editing. pSPN comprises a 1 O2 -generating backbone grafted with polyethylenimine brushes through 1 O2 -cleavable linkers. NIR photoirradiation spontaneously triggers the cleavage of gene vectors from pSPN, resulting in the release of CRISPR/Cas9 plasmids and subsequently initiating gene editing. This system affords 15- and 1.8-fold enhancement in repaired gene expression relative to the nonirradiated controls in living cells and mice, respectively. As this approach does not require any specific modifications on biomolecular components, pSPN represents the first generic nanotransducer for in vivo regulation of CRISPR/Cas9 gene editing.},
}

Animals
*CRISPR-Cas Systems
Female
Fluorescence
Gene Editing/*methods
Gene Transfer Techniques
Genetic Vectors
HeLa Cells
Humans
Mice, Nude
Nanostructures/*chemistry
Photochemical Processes
Plasmids/genetics
Polyethylene Glycols/chemistry
Polyethyleneimine/chemistry
Polymers/*chemistry
Proof of Concept Study
Singlet Oxygen

@article {pmid30791200,
year = {2020},
author = {Zhou, J and Li, D and Wang, G and Wang, F and Kunjal, M and Joldersma, D and Liu, Z},
title = {Application and future perspective of CRISPR/Cas9 genome editing in fruit crops.},
journal = {Journal of integrative plant biology},
volume = {62},
number = {3},
pages = {269-286},
doi = {10.1111/jipb.12793},
pmid = {30791200},
issn = {1744-7909},
support = {1444987//NSF/ ; 11889048//USDA/ ; //Maryland Agricultural Experiment Station Hatch Project/ ; 201606320096//Ministry of Education of China/ ; 201706850061//Ministry of Education of China/ ; T32 GM008553/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics/*metabolism ; Fruit/genetics/*metabolism ; Gene Editing/*methods ; Plants, Genetically Modified/genetics/*metabolism ; },
abstract = {Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer-friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.},
}

Alleles
CRISPR-Cas Systems/genetics
Crops, Agricultural/genetics/*metabolism
Fruit/genetics/*metabolism
Gene Editing/*methods
Plants, Genetically Modified/genetics/*metabolism

@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},
doi = {10.3389/fbioe.2020.589468},
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},
doi = {10.3389/fmicb.2020.594737},
pmid = {33193271},
issn = {1664-302X},
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},
doi = {10.3389/fmicb.2020.593700},
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},
doi = {10.3389/fnins.2020.579062},
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 {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 {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 & 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 = {},
doi = {10.3390/v12111286},
pmid = {33182769},
issn = {1999-4915},
support = {773567//European Union's Horizon H2020 research and innovation programme/ ; },
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 {pmid33152914,
year = {2020},
author = {Lotfi, M and Rezaei, N},
title = {CRISPR/Cas13: A potential therapeutic option of COVID-19.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {131},
number = {},
pages = {110738},
pmid = {33152914},
issn = {1950-6007},
mesh = {Betacoronavirus/drug effects/*genetics ; 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 & inhibitors/*genetics ; },
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.},
}

Betacoronavirus/drug effects/*genetics
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 & inhibitors/*genetics

@article {pmid32156257,
year = {2020},
author = {Berry, LK and Thomas, GH and Thorpe, PH},
title = {CATS: Cas9-assisted tag switching. A high-throughput method for exchanging genomic peptide tags in yeast.},
journal = {BMC genomics},
volume = {21},
number = {1},
pages = {221},
pmid = {32156257},
issn = {1471-2164},
support = {FC001003//Francis Crick Institute/ ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Gene Editing ; Green Fluorescent Proteins/*genetics/metabolism ; High-Throughput Screening Assays ; Peptides/*genetics/metabolism ; Plasmids/genetics ; RNA, Guide/genetics ; Saccharomyces cerevisiae/genetics/*growth & development ; Staining and Labeling ; },
abstract = {BACKGROUND: The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library.

RESULTS: We present a new recombination-based method, CATS - Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining.

CONCLUSIONS: The method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.},
}

CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems
Gene Editing
Green Fluorescent Proteins/*genetics/metabolism
High-Throughput Screening Assays
Peptides/*genetics/metabolism
Plasmids/genetics
RNA, Guide/genetics
Saccharomyces cerevisiae/genetics/*growth & development
Staining and Labeling

@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 = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.0c03412},
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 = {},
number = {},
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 "predict" infection outcomes to "decide" 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 "interests" 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},
doi = {10.3389/fmicb.2020.576673},
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 {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 = {},
doi = {10.3390/biom10111523},
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 {pmid32694110,
year = {2020},
author = {Qin, RY and Wei, PC},
title = {[Prime editing creates a novel dimension of plant precise genome editing].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {6},
pages = {519-523},
doi = {10.16288/j.yczz.20-125},
pmid = {32694110},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *Gene Editing ; Genome, Plant/genetics ; *Oryza/genetics ; },
abstract = {The precise genome editing has not been well established in plants, largely because of the limited frequency of homology recombination and the delivery barrier of donor templates. Recently, Dr. Caixia Gao's group from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, developed a series of plant prime editors (PPEs), which mediats the prime editing in the genomes of rice and wheat. The PPE systems are able to generate all 12 kinds of programmable base substitutions, as well desired multiplex nucleotide substitutions and small deletions or insertions without DNA double-strand breaks, thus providing versatile tools for precise plant genome editing. Herein, we introduce the structure and the editing capacity of the PPEs. The attemp on efficiency enhancements of PPEs and other PPEs are also discussed, which may provide a reference for appropriate application of PPEs in plants and also for continuous optimization of the editing tools.},
}

*CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded
*Gene Editing
Genome, Plant/genetics
*Oryza/genetics

@article {pmid32298553,
year = {2020},
author = {Leonova, EI and Gainetdinov, RR},
title = {CRISPR/Cas9 Technology in Translational Biomedicine.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {54},
number = {3},
pages = {354-370},
doi = {10.33594/000000224},
pmid = {32298553},
issn = {1421-9778},
support = {N 19-75-30008//Russian Science Foundation/Russia ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics/physiology ; Caspase 9/chemistry/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; Disease Models, Animal ; Gene Editing/*methods ; Gene Expression Regulation/*genetics ; Genetic Engineering/methods ; Genetic Therapy/*methods ; Homologous Recombination/genetics ; Humans ; RNA, Guide/genetics ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - RNA-guided Cas9 endonuclease system has provided a fast and efficient method for precise genome editing in diverse mammalian species, including humans. The CRISPR/Cas9 technology allows generation of modifications into site-specific locations of the selected genes in one major step by carrying deletions, insertions or DNA donor-directed precise sequence modifications. Cas9 forms a nucleoprotein complex with a sequence-specific guide RNA to create double-stranded breaks in complementary DNA target. Further, double-stranded break repair machinery leads to the intended gene modifications. The CRISPR/Cas9 system is widely used technique for genome modification, editing and other biotechnology applications, such as functional annotation, a system for visualization of specific genomic loci and transcriptional control of genes. CRISPR/Cas9-mediated manipulation of the laboratory animal genomes has contributed to the understanding of gene functions and has become a popular approach for modeling human disorders. Furthermore, the growing application of CRISPR-Cas9 system to human genes emerges as an extremely powerful technology for the molecular characterization and treatment of human disease. In this review we present the essential principles of CRISPR/Cas9 technology and the recent advances in its use in translational biomedicine.},
}

Animals
CRISPR-Cas Systems/*genetics/physiology
Caspase 9/chemistry/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
DNA Breaks, Double-Stranded
DNA End-Joining Repair/genetics
Disease Models, Animal
Gene Editing/*methods
Gene Expression Regulation/*genetics
Genetic Engineering/methods
Genetic Therapy/*methods
Homologous Recombination/genetics
Humans
RNA, Guide/genetics

@article {pmid32284553,
year = {2020},
author = {Kaminski, MM and Alcantar, MA and Lape, IT and Greensmith, R and Huske, AC and Valeri, JA and Marty, FM and Klämbt, V and Azzi, J and Akalin, E and Riella, LV and Collins, JJ},
title = {A CRISPR-based assay for the detection of opportunistic infections post-transplantation and for the monitoring of transplant rejection.},
journal = {Nature biomedical engineering},
volume = {4},
number = {6},
pages = {601-609},
doi = {10.1038/s41551-020-0546-5},
pmid = {32284553},
issn = {2157-846X},
mesh = {Biomarkers/blood/urine ; *CRISPR-Cas Systems ; Chemokine CXCL9/blood/urine ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytomegalovirus/genetics/isolation & purification ; Cytomegalovirus Infections/diagnosis ; DNA, Viral/blood/genetics/urine ; Graft Rejection/*virology ; Humans ; Kidney ; Kidney Diseases/virology ; Kidney Transplantation/adverse effects ; Male ; Middle Aged ; Opportunistic Infections/*diagnosis ; Pathology, Molecular/*methods ; Point-of-Care Testing ; Polyomavirus/genetics/isolation & purification ; Polyomavirus Infections/diagnosis ; Postoperative Complications/diagnosis ; RNA, Messenger ; Tumor Virus Infections/diagnosis ; },
abstract = {In organ transplantation, infection and rejection are major causes of graft loss. They are linked by the net state of immunosuppression. To diagnose and treat these conditions earlier, and to improve long-term patient outcomes, refined strategies for the monitoring of patients after graft transplantation are needed. Here, we show that a fast and inexpensive assay based on CRISPR-Cas13 accurately detects BK polyomavirus DNA and cytomegalovirus DNA from patient-derived blood and urine samples, as well as CXCL9 messenger RNA (a marker of graft rejection) at elevated levels in urine samples from patients experiencing acute kidney transplant rejection. The assay, which we adapted for lateral-flow readout, enables-via simple visualization-the post-transplantation monitoring of common opportunistic viral infections and of graft rejection, and should facilitate point-of-care post-transplantation monitoring.},
}

Biomarkers/blood/urine
*CRISPR-Cas Systems
Chemokine CXCL9/blood/urine
Clustered Regularly Interspaced Short Palindromic Repeats
Cytomegalovirus/genetics/isolation & purification
Cytomegalovirus Infections/diagnosis
DNA, Viral/blood/genetics/urine
Graft Rejection/*virology
Humans
Kidney
Kidney Diseases/virology
Kidney Transplantation/adverse effects
Male
Middle Aged
Opportunistic Infections/*diagnosis
Pathology, Molecular/*methods
Point-of-Care Testing
Polyomavirus/genetics/isolation & purification
Polyomavirus Infections/diagnosis
Postoperative Complications/diagnosis
RNA, Messenger
Tumor Virus Infections/diagnosis

@article {pmid32278427,
year = {2020},
author = {Zhang, M and Liu, C and Shi, Y and Wu, J and Wu, J and Chen, H},
title = {Selective endpoint visualized detection of Vibrio parahaemolyticus with CRISPR/Cas12a assisted PCR using thermal cycler for on-site application.},
journal = {Talanta},
volume = {214},
number = {},
pages = {120818},
doi = {10.1016/j.talanta.2020.120818},
pmid = {32278427},
issn = {1873-3573},
mesh = {Animals ; Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; Electrochemical Techniques ; Food Contamination/analysis ; Particle Size ; Penaeidae/*microbiology ; *Polymerase Chain Reaction ; RNA, Bacterial/*genetics ; Surface Properties ; *Temperature ; Vibrio parahaemolyticus/genetics/*isolation & purification ; },
abstract = {Vibrio parahaemolyticus is a major cause of seafood-associated food poisoning. It is of great significance to develop an accurate, simple and cost-effective method to identify infected seafood, especially for on-site application. Polymerase chain reaction (PCR) remains the golden standard for nucleic acid detection. But traditional methods heavily reply on sophisticated instrument and specialized operators, which limits the application for on-site detections. Here we developed a novel, specific and visualized detection method for PCR based on CRISPR/Cas12a system. On a low-cost thermal cycler, amplification reaction can be conducted easily. The CRISPR/Cas12a system was specifically designed to evaluate amplicons, eliminating false positive results. Besides the negative samples remained colorless, the positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye using a homemade UV device. The presented detection method was verified by detecting shrimp samples. The limit of detection is 1.02 × 102 copies/μL. This presented method provided a new strategy for specific endpoint detection of PCR and advanced its application in field for food safety assurance.},
}

Animals
Biosensing Techniques
CRISPR-Cas Systems/*genetics
Electrochemical Techniques
Food Contamination/analysis
Particle Size
Penaeidae/*microbiology
*Polymerase Chain Reaction
RNA, Bacterial/*genetics
Surface Properties
*Temperature
Vibrio parahaemolyticus/genetics/*isolation & purification

@article {pmid32181354,
year = {2020},
author = {Champer, SE and Oh, SY and Liu, C and Wen, Z and Clark, AG and Messer, PW and Champer, J},
title = {Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs.},
journal = {Science advances},
volume = {6},
number = {10},
pages = {eaaz0525},
pmid = {32181354},
issn = {2375-2548},
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 ; Anopheles/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drosophila melanogaster/*genetics/metabolism ; Female ; Gene Drive Technology/*methods ; Gene Editing/methods ; Male ; Models, Genetic ; RNA, Guide/*genetics/metabolism ; },
abstract = {The rapid evolution of resistance alleles poses a major obstacle for genetic manipulation of populations with CRISPR homing gene drives. One proposed solution is using multiple guide RNAs (gRNAs), allowing a drive to function even if some resistant target sites are present. Here, we develop a model of homing mechanisms parameterized by experimental studies. Our model incorporates several factors affecting drives with multiple gRNAs, including timing of cleavage, reduction in homology-directed repair efficiency due to imperfect homology, Cas9 activity saturation, gRNA activity level variance, and incomplete homology-directed repair. We find that homing drives have an optimal number of gRNAs, usually between two and eight, depending on the specific drive type and performance parameters. These results contradict the notion that resistance rates can be reduced to arbitrarily low levels by gRNA multiplexing and highlight the need for combined approaches to counter resistance evolution in CRISPR homing drives.},
}

Alleles
Animals
Anopheles/*genetics/metabolism
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Drosophila melanogaster/*genetics/metabolism
Female
Gene Drive Technology/*methods
Gene Editing/methods
Male
Models, Genetic
RNA, Guide/*genetics/metabolism

@article {pmid32176842,
year = {2020},
author = {Bhardwaj, R and Augustynek, BS and Ercan-Herbst, E and Kandasamy, P and Seedorf, M and Peinelt, C and Hediger, MA},
title = {Ca2+/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca2+-Dependent Inactivation of the Orai1 Channel.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {54},
number = {2},
pages = {252-270},
doi = {10.33594/000000218},
pmid = {32176842},
issn = {1421-9778},
support = {CRSII5_180326, CRSII3_160782//Schweizerischer Nationalfonds (SNF), Sinergia Grant/Switzerland ; },
mesh = {CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium Channels/genetics/*metabolism ; Calcium Signaling ; Calmodulin/*metabolism ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Hydrophobic and Hydrophilic Interactions ; Membrane Proteins/metabolism ; Models, Chemical ; Models, Molecular ; Mutation ; Neoplasm Proteins/*chemistry/genetics/metabolism/*physiology ; ORAI1 Protein/chemistry/genetics/*metabolism ; Protein Binding ; Protein Domains ; Stromal Interaction Molecule 1/*chemistry/genetics/metabolism/*physiology ; Up-Regulation ; },
abstract = {BACKGROUND/AIMS: Store-operated Ca2+ entry (SOCE) through plasma membrane Ca2+ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca2+ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca2+-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca2+ release-activated Ca2+ current (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca2+/calmodulin (Ca2+/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca2+/CaM-mediated SCDI of ICRAC is as yet unclear.

METHODS: The Ca2+/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca2+ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca2+/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology.

RESULTS: Here we identified residues Leu374/Val375 (H1) and Leu390/Phe391 (H2) within SOAR that serve as hydrophobic anchor sites for Ca2+/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca2+/CaM binding. Single residue mutations of Phe391 to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca2+/CaM. Hence, the role of H2 residues in Ca2+/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca2+/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI.

CONCLUSION: Taken together, we propose a mechanistic model, wherein binding of Ca2+/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca2+/CaM are functionally coordinated to control ICRAC.},
}

CRISPR-Cas Systems
Calcium/*metabolism
Calcium Channels/genetics/*metabolism
Calcium Signaling
Calmodulin/*metabolism
Gene Knockout Techniques
HEK293 Cells
Humans
Hydrophobic and Hydrophilic Interactions
Membrane Proteins/metabolism
Models, Chemical
Models, Molecular
Mutation
Neoplasm Proteins/*chemistry/genetics/metabolism/*physiology
ORAI1 Protein/chemistry/genetics/*metabolism
Protein Binding
Protein Domains
Stromal Interaction Molecule 1/*chemistry/genetics/metabolism/*physiology
Up-Regulation

@article {pmid32176542,
year = {2020},
author = {Feng, W and Liu, C and Spinozzi, S and Wang, L and Evans, SM and Chen, J},
title = {Identifying the Cardiac Dyad Proteome In Vivo by a BioID2 Knock-In Strategy.},
journal = {Circulation},
volume = {141},
number = {11},
pages = {940-942},
pmid = {32176542},
issn = {1524-4539},
support = {R01 HL130295/HL/NHLBI NIH HHS/United States ; R01 HL137957/HL/NHLBI NIH HHS/United States ; R01 HL144872/HL/NHLBI NIH HHS/United States ; R01 HL146759/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Biotinylation ; CRISPR-Cas Systems ; Excitation Contraction Coupling/*genetics ; Gene Editing ; Gene Knock-In Techniques/*methods ; Gene Ontology ; Membrane Proteins/genetics/metabolism/*physiology ; Mice ; Muscle Proteins/genetics/metabolism/*physiology ; Myocytes, Cardiac/*physiology ; Oligopeptides ; Protein Interaction Mapping ; *Proteome ; Recombinant Fusion Proteins/metabolism ; Sarcoplasmic Reticulum/metabolism ; Tandem Mass Spectrometry ; },
}

Animals
Biotinylation
CRISPR-Cas Systems
Excitation Contraction Coupling/*genetics
Gene Editing
Gene Knock-In Techniques/*methods
Gene Ontology
Membrane Proteins/genetics/metabolism/*physiology
Mice
Muscle Proteins/genetics/metabolism/*physiology
Myocytes, Cardiac/*physiology
Oligopeptides
Protein Interaction Mapping
*Proteome
Recombinant Fusion Proteins/metabolism
Sarcoplasmic Reticulum/metabolism
Tandem Mass Spectrometry

@article {pmid32095520,
year = {2020},
author = {Wang, L and Yang, Y and Breton, C and Bell, P and Li, M and Zhang, J and Che, Y and Saveliev, A and He, Z and White, J and Latshaw, C and Xu, C and McMenamin, D and Yu, H and Morizono, H and Batshaw, ML and Wilson, JM},
title = {A mutation-independent CRISPR-Cas9-mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency.},
journal = {Science advances},
volume = {6},
number = {7},
pages = {eaax5701},
pmid = {32095520},
issn = {2375-2548},
support = {P01 HD057247/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA Repair/genetics ; Dependovirus/genetics ; Dietary Proteins ; Disease Models, Animal ; *Gene Targeting ; Genetic Loci ; *Genetic Therapy ; Genetic Vectors/metabolism ; INDEL Mutation/genetics ; Liver/enzymology/pathology ; Male ; Mice ; Mutation/*genetics ; Ornithine Carbamoyltransferase/genetics/metabolism ; Ornithine Carbamoyltransferase Deficiency Disease/*genetics/*therapy ; Time Factors ; },
abstract = {Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality. Although a promising treatment for late-onset OTC deficiency, adeno-associated virus (AAV) neonatal gene therapy would only provide short-term therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation. CRISPR-Cas9-mediated homology-directed repair can correct a G-to-A mutation in 10% of OTC alleles in the livers of newborn OTC spfash mice. However, an editing vector able to correct one mutation would not be applicable for patients carrying different OTC mutations, plus expression would not be fast enough to treat a hyperammonemia crisis. Here, we describe a dual-AAV vector system that accomplishes rapid short-term expression from a non-integrated minigene and long-term expression from the site-specific integration of this minigene without any selective growth advantage for OTC-positive cells in newborns. This CRISPR-Cas9 gene-targeting approach may be applicable to all patients with OTC deficiency, irrespective of mutation and/or clinical state.},
}

Animals
CRISPR-Cas Systems/*genetics
DNA Repair/genetics
Dependovirus/genetics
Dietary Proteins
Disease Models, Animal
*Gene Targeting
Genetic Loci
*Genetic Therapy
Genetic Vectors/metabolism
INDEL Mutation/genetics
Liver/enzymology/pathology
Male
Mice
Mutation/*genetics
Ornithine Carbamoyltransferase/genetics/metabolism
Ornithine Carbamoyltransferase Deficiency Disease/*genetics/*therapy
Time Factors

@article {pmid32095517,
year = {2020},
author = {Skryabin, BV and Kummerfeld, DM and Gubar, L and Seeger, B and Kaiser, H and Stegemann, A and Roth, J and Meuth, SG and Pavenstädt, H and Sherwood, J and Pap, T and Wedlich-Söldner, R and Sunderkötter, C and Schwartz, YB and Brosius, J and Rozhdestvensky, TS},
title = {Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9-mediated genome editing events.},
journal = {Science advances},
volume = {6},
number = {7},
pages = {eaax2941},
pmid = {32095517},
issn = {2375-2548},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Crosses, Genetic ; DNA/*genetics ; Female ; *Gene Editing ; Gene Targeting ; Genetic Loci ; Genome ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Models, Animal ; *Templates, Genetic ; },
abstract = {CRISPR-Cas9-mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.},
}

Animals
CRISPR-Cas Systems/*genetics
Crosses, Genetic
DNA/*genetics
Female
*Gene Editing
Gene Targeting
Genetic Loci
Genome
Male
Mice, Inbred C57BL
Mice, Knockout
Models, Animal
*Templates, Genetic

@article {pmid32050051,
year = {2020},
author = {Hou, C and Yang, Y and Xing, Y and Zhan, C and Liu, G and Liu, X and Liu, C and Zhan, J and Xu, D and Bai, Z},
title = {Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology.},
journal = {Yeast (Chichester, England)},
volume = {37},
number = {4},
pages = {305-312},
doi = {10.1002/yea.3462},
pmid = {32050051},
issn = {1097-0061},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Culture Media/chemistry ; Fungal Proteins/*genetics/metabolism ; Gene Editing ; Gene Expression Regulation, Fungal ; Methanol/metabolism ; Pichia/*genetics/metabolism ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide ; Transcription Factors ; },
abstract = {A highly efficient and targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system was constructed for Pichia pastoris (syn Komagataella phaffii). Plasmids containing single guide RNA and the methanol expression regulator 1 (MXR1) homology arms were used to precisely edit the transcriptional activator Mxr1 on the P. pastoris genome. At the S215 amino acid position of Mxr1, one, two, and three nucleotides were precisely deleted or inserted, and S215 was also mutated to S215A via a single-base substitution. Sequencing of polymerase chain reaction (PCR) amplicons in the region spanning MXR1 showed that CRISPR/Cas9 technology enabled efficient and precise gene editing of P. pastoris. The expression levels of several of the Mxr1-targeted genes, AOX1, AOX2, DAS1, and DAS2, in strains containing the various mutated variants of MXR1, were then detected through reverse transcription PCR following induction in methanol-containing culture medium. The frameshift mutations of Mxr1 led to almost zero transcription of AOX1, DAS1, and DAS2, while that of AOX2 was reduced to 60%. For the Mxr1 S215A mutant, the transcription of AOX1, AOX2, DAS1, and DAS2 was also reduced by nearly 60%. Based on these results, it is apparent that the transcription of AOX1, DAS1, and DAS2 is exclusively regulated by Mxr1 and serine phosphorylation at Mxr1 residue 215 is not critical for this function. In contrast, the transcription of AOX2 is mainly dependent on the phosphorylation of this residue. CRISPR/Cas9 technology was, therefore, successfully applied to the targeted editing of MXR1 on the P. pastoris genome, and it provided an effective method for the study of this transcription factor and its targets.},
}

Base Sequence
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Culture Media/chemistry
Fungal Proteins/*genetics/metabolism
Gene Editing
Gene Expression Regulation, Fungal
Methanol/metabolism
Pichia/*genetics/metabolism
Plasmids/genetics/metabolism
Promoter Regions, Genetic
RNA, Guide
Transcription Factors

@article {pmid31885035,
year = {2019},
author = {Hasan, A and Yeom, HS and Ryu, J and Bode, HB and Kim, Y},
title = {Phenylethylamides derived from bacterial secondary metabolites specifically inhibit an insect serotonin receptor.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {20358},
pmid = {31885035},
issn = {2045-2322},
mesh = {Amino Acid Sequence ; Animals ; Bacteria/classification/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression ; Hemocytes/metabolism ; Immunity, Cellular ; Insect Proteins/*antagonists & inhibitors ; Larva ; Phagocytosis/genetics/immunology ; Phenethylamines/chemistry/*pharmacology ; Phylogeny ; RNA Interference ; Receptors, Serotonin/genetics/*metabolism ; Secondary Metabolism ; Serotonin Antagonists/chemistry/*pharmacology ; },
abstract = {Serotonin (5-hydroxytryptamine: 5-HT) is a biogenic monoamine that mediates immune responses and modulates nerve signal in insects. Se-5HTR, a specific receptor of serotonin, has been identified in the beet armyworm, Spodoptera exigua. It is classified into subtype 7 among known 5HTRs. Se-5HTR was expressed in all developmental stages of S. exigua. It was expressed in all tested tissues of larval stage. Its expression was up-regulated in hemocytes and fat body in response to immune challenge. RNA interference (RNAi) of Se-5HTR exhibited significant immunosuppression by preventing cellular immune responses such as phagocytosis and nodulation. Treatment with an inhibitor (SB-269970) specific to 5HTR subtype 7 resulted in significant immunosuppression. Furthermore, knockout mutant of Se-5HTR by CRISPR-Cas9 led to significant reduction of phagocytotic activity of S. exigua hemocytes. Such immunosuppression was also induced by bacterial secondary metabolites derived from Xenorhabdus and Photorhabdus. To determine specific bacterial metabolites inhibiting Se-5HTR, this study screened 37 bacterial secondary metabolites with respect to cellular immune responses associated with Se-5HTR and selected 10 potent inhibitors. These 10 selected compounds competitively inhibited cellular immune responses against 5-HT and shared phenylethylamide (PEA) chemical skeleton. Subsequently, 46 PEA derivatives were screened and resulting potent chemicals were used to design a compound to be highly inhibitory against Se-5HTR. The designed compound was chemically synthesized. It showed high immunosuppressive activities along with specific and competitive inhibition activity for Se-5HTR. This study reports the first 5HT receptor from S. exigua and provides its specific inhibitor designed from bacterial metabolites and their derivatives.},
}

Amino Acid Sequence
Animals
Bacteria/classification/genetics/metabolism
CRISPR-Cas Systems
Gene Expression
Hemocytes/metabolism
Immunity, Cellular
Insect Proteins/*antagonists & inhibitors
Larva
Phagocytosis/genetics/immunology
Phenethylamines/chemistry/*pharmacology
Phylogeny
RNA Interference
Receptors, Serotonin/genetics/*metabolism
Secondary Metabolism
Serotonin Antagonists/chemistry/*pharmacology

@article {pmid31811188,
year = {2019},
author = {Santos, JM and Oudinet, C and Schöne, L and Dauba, A and Khamlichi, AA},
title = {Essential role of the initial activation signal in isotype selection upon deletion of a transcriptionally committed promoter.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18543},
pmid = {31811188},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Immunoglobulin Class Switching/*genetics ; Immunoglobulin Heavy Chains/*genetics ; Immunoglobulin Isotypes/*genetics ; Mice ; Promoter Regions, Genetic/genetics ; *Recombination, Genetic ; Sequence Deletion ; },
abstract = {Class switch recombination (CSR), which targets exclusively the constant region of the immunoglobulin heavy chain (IgH) locus, plays an important role in humoral immunity by generating different antibody effector functions. The IgH constant locus contains multiple genes controlled by isotype (I) promoters induced by extracellular signals that activate specific I promoters, leading to B cell commitment. However, it is unknown whether after initial commitment to one promoter, non-responsive I promoters are irreversibly silent or if they can be activated after exposure to their specific inducers. Here, we studied the murine cell line CH12, which is committed to produce IgA in response to TGF-β. We show that, although other promoters than Iα are transcriptionally inactive, they are not irreversibly silent. Following deletion of the committed Iα promoter by CRISPR/Cas9, other I promoters display a complex transcriptional pattern largely dependent on the initial committing signal.},
}

Animals
CRISPR-Cas Systems
Cell Line, Tumor
Gene Editing
Immunoglobulin Class Switching/*genetics
Immunoglobulin Heavy Chains/*genetics
Immunoglobulin Isotypes/*genetics
Mice
Promoter Regions, Genetic/genetics
*Recombination, Genetic
Sequence Deletion

@article {pmid31792362,
year = {2019},
author = {Leinonen, JT and Chen, YC and Pennonen, J and Lehtonen, L and Junna, N and Tukiainen, T and Panula, P and Widén, E},
title = {LIN28B affects gene expression at the hypothalamic-pituitary axis and serum testosterone levels.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18060},
pmid = {31792362},
issn = {2045-2322},
support = {MC_PC_12028/MRC_/Medical Research Council/United Kingdom ; MC_PC_17228/MRC_/Medical Research Council/United Kingdom ; MC_QA137853/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Alleles ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Computational Biology ; Datasets as Topic ; Estrogen Receptor alpha/metabolism ; Female ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Humans ; Hypothalamo-Hypophyseal System/*metabolism ; Hypothalamus/metabolism ; Male ; Models, Animal ; Pituitary Gland/metabolism ; Polymorphism, Single Nucleotide ; Pro-Opiomelanocortin/*metabolism ; RNA-Binding Proteins/*genetics/*metabolism ; RNA-Seq ; Sexual Maturation/genetics ; Testosterone/*blood/metabolism ; Zebrafish ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {Genome-wide association studies (GWAS) have recurrently associated sequence variation nearby LIN28B with pubertal timing, growth and disease. However, the biology linking LIN28B with these traits is still poorly understood. With our study, we sought to elucidate the mechanisms behind the LIN28B associations, with a special focus on studying LIN28B function at the hypothalamic-pituitary (HP) axis that is ultimately responsible for pubertal onset. Using CRISPR-Cas9 technology, we first generated lin28b knockout (KO) zebrafish. Compared to controls, the lin28b KO fish showed both accelerated growth tempo, reduced adult size and increased expression of mitochondrial genes during larval development. Importantly, data from the knockout zebrafish models and adult humans imply that LIN28B expression has potential to affect gene expression in the HP axis. Specifically, our results suggest that LIN28B expression correlates positively with the expression of ESR1 in the hypothalamus and POMC in the pituitary. Moreover, we show how the pubertal timing advancing allele (T) for rs7759938 at the LIN28B locus associates with higher testosterone levels in the UK Biobank data. Overall, we provide novel evidence that LIN28B contributes to the regulation of sex hormone pathways, which might help explain why the gene associates with several distinct traits.},
}

Alleles
Animals
Animals, Genetically Modified
CRISPR-Cas Systems/genetics
Computational Biology
Datasets as Topic
Estrogen Receptor alpha/metabolism
Female
*Gene Expression Regulation, Developmental
Gene Knockdown Techniques
Gene Knockout Techniques
Humans
Hypothalamo-Hypophyseal System/*metabolism
Hypothalamus/metabolism
Male
Models, Animal
Pituitary Gland/metabolism
Polymorphism, Single Nucleotide
Pro-Opiomelanocortin/*metabolism
RNA-Binding Proteins/*genetics/*metabolism
RNA-Seq
Sexual Maturation/genetics
Testosterone/*blood/metabolism
Zebrafish
Zebrafish Proteins/genetics/*metabolism

@article {pmid31792264,
year = {2019},
author = {Munawar, U and Roth, M and Barrio, S and Wajant, H and Siegmund, D and Bargou, RC and Kortüm, KM and Stühmer, T},
title = {Assessment of TP53 lesions for p53 system functionality and drug resistance in multiple myeloma using an isogenic cell line model.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {18062},
pmid = {31792264},
issn = {2045-2322},
mesh = {Alleles ; Antineoplastic Agents, Alkylating/*pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*genetics ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Heterozygote ; Homozygote ; Humans ; Melphalan/pharmacology/therapeutic use ; Multiple Myeloma/drug therapy/*genetics/pathology ; Point Mutation ; Signal Transduction/genetics ; Tumor Suppressor Protein p53/*genetics/metabolism ; },
abstract = {Recent advances in molecular diagnostics have shown that lesions affecting both copies of the gene for tumor suppressor protein 53 (TP53) count among the most powerful predictors for high-risk disease in multiple myeloma (MM). However, the functional relevance and potential therapeutic implications of single hits to TP53 remain less well understood. Here, we have for the first time approximated the different constellations of mono- and bi-allelic TP53 lesions observed in MM patients within the frame of a single MM cell line model and assessed their potential to disrupt p53 system functionality and to impart drug resistance. Both types of common first hit: point mutation with expression of mutant p53 protein or complete loss of contribution from one of two wildtype alleles strongly impaired p53 system functionality and increased resistance to melphalan. Second hits abolished remaining p53 activity and increased resistance to genotoxic drugs even further. These results fit well with the clinical drive to TP53 single- and double-hit disease in MM patients, provide a rationale for the most commonly observed double-hit constellation (del17p+ TP53 point mutation), and underscore the potential increases in MM cell malignancy associated with any type of initial TP53 lesion.},
}

Alleles
Antineoplastic Agents, Alkylating/*pharmacology/therapeutic use
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Drug Resistance, Neoplasm/*genetics
Gene Editing
*Gene Expression Regulation, Neoplastic
Heterozygote
Homozygote
Humans
Melphalan/pharmacology/therapeutic use
Multiple Myeloma/drug therapy/*genetics/pathology
Point Mutation
Signal Transduction/genetics
Tumor Suppressor Protein p53/*genetics/metabolism

@article {pmid31748650,
year = {2019},
author = {Huang, J and Chen, M and Xu, ES and Luo, L and Ma, Y and Huang, W and Floyd, W and Klann, TS and Kim, SY and Gersbach, CA and Cardona, DM and Kirsch, DG},
title = {Genome-wide CRISPR Screen to Identify Genes that Suppress Transformation in the Presence of Endogenous KrasG12D.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17220},
pmid = {31748650},
issn = {2045-2322},
support = {T32 GM007171/GM/NIGMS NIH HHS/United States ; T32GM007171//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/International ; R35CA197616//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/International ; F30CA206424//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Proliferation ; Cell Transformation, Neoplastic/genetics/metabolism/*pathology ; Cells, Cultured ; Embryo, Mammalian/cytology/metabolism ; Fibroblasts/cytology/metabolism ; Gene Expression Regulation, Neoplastic ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; *Mutation ; Neoplasm Proteins/*antagonists & inhibitors/genetics ; Proto-Oncogene Proteins p21(ras)/*genetics ; Sarcoma, Experimental/genetics/pathology/*prevention & control ; Signal Transduction ; },
abstract = {Cooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.},
}

Animals
CRISPR-Cas Systems
*Cell Proliferation
Cell Transformation, Neoplastic/genetics/metabolism/*pathology
Cells, Cultured
Embryo, Mammalian/cytology/metabolism
Fibroblasts/cytology/metabolism
Gene Expression Regulation, Neoplastic
Mice
Mice, Inbred C57BL
Mice, Nude
*Mutation
Neoplasm Proteins/*antagonists & inhibitors/genetics
Proto-Oncogene Proteins p21(ras)/*genetics
Sarcoma, Experimental/genetics/pathology/*prevention & control
Signal Transduction

@article {pmid31748579,
year = {2019},
author = {Lee, JY and Dominguez, AA and Nam, S and Stowers, RS and Qi, LS and Chaudhuri, O},
title = {Identification of cell context-dependent YAP-associated proteins reveals β1 and β4 integrin mediate YAP translocation independently of cell spreading.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17188},
pmid = {31748579},
issn = {2045-2322},
support = {P30 CA124435/CA/NCI NIH HHS/United States ; R37 CA214136/CA/NCI NIH HHS/United States ; S10 RR027425/RR/NCRR NIH HHS/United States ; },
mesh = {Actin Cytoskeleton/*metabolism ; Actins/*metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cells, Cultured ; Extracellular Matrix/*metabolism ; Humans ; Integrin beta1/chemistry/genetics/*metabolism ; Integrin beta4/chemistry/genetics/*metabolism ; *Mechanotransduction, Cellular ; Phosphorylation ; Protein Processing, Post-Translational ; Protein Transport ; Signal Transduction ; Transcription Factors/genetics/*metabolism ; },
abstract = {Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with β1 integrin under activating conditions, and β4 integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of β1 integrin (ΔITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. ΔITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, β4 integrin KO (ΔITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.},
}

Actin Cytoskeleton/*metabolism
Actins/*metabolism
CRISPR-Cas Systems
Cell Cycle Proteins/genetics/*metabolism
Cells, Cultured
Extracellular Matrix/*metabolism
Humans
Integrin beta1/chemistry/genetics/*metabolism
Integrin beta4/chemistry/genetics/*metabolism
*Mechanotransduction, Cellular
Phosphorylation
Protein Processing, Post-Translational
Protein Transport
Signal Transduction
Transcription Factors/genetics/*metabolism

@article {pmid31745156,
year = {2019},
author = {Hooghvorst, I and López-Cristoffanini, C and Nogués, S},
title = {Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17077},
pmid = {31745156},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cucurbitaceae/*genetics/growth & development/metabolism ; *Gene Editing ; Genome, Plant ; *Mutation ; Oxidoreductases/*antagonists & inhibitors/genetics/metabolism ; Plant Proteins/*antagonists & inhibitors/genetics/metabolism ; Plants, Genetically Modified/*genetics/growth & development/metabolism ; },
abstract = {CRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42-45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.},
}

*CRISPR-Cas Systems
Cucurbitaceae/*genetics/growth & development/metabolism
*Gene Editing
Genome, Plant
*Mutation
Oxidoreductases/*antagonists & inhibitors/genetics/metabolism
Plant Proteins/*antagonists & inhibitors/genetics/metabolism
Plants, Genetically Modified/*genetics/growth & development/metabolism

@article {pmid31745112,
year = {2019},
author = {Sullivan, NT and Dampier, W and Chung, CH and Allen, AG and Atkins, A and Pirrone, V and Homan, G and Passic, S and Williams, J and Zhong, W and Kercher, K and Desimone, M and Li, L and C Antell, G and Mell, JC and Ehrlich, GD and Szep, Z and Jacobson, JM and Nonnemacher, MR and Wigdahl, B},
title = {Novel gRNA design pipeline to develop broad-spectrum CRISPR/Cas9 gRNAs for safe targeting of the HIV-1 quasispecies in patients.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {17088},
pmid = {31745112},
issn = {2045-2322},
support = {T32 MH079785/MH/NIMH NIH HHS/United States ; P30 MH092177/MH/NIMH NIH HHS/United States ; R01 MH110360/MH/NIMH NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cohort Studies ; Computational Biology ; Female ; *Gene Editing ; Genome, Viral ; HIV Infections/*genetics/virology ; HIV Long Terminal Repeat/genetics ; HIV-1/*genetics/*growth & development/metabolism ; Humans ; Male ; Middle Aged ; Proviruses/*genetics ; Quasispecies/*genetics ; RNA, Guide/*genetics ; },
abstract = {The CRISPR/Cas9 system has been proposed as a cure strategy for HIV. However, few published guide RNAs (gRNAs) are predicted to cleave the majority of HIV-1 viral quasispecies (vQS) observed within and among patients. We report the design of a novel pipeline to identify gRNAs that target HIV across a large number of infected individuals. Next generation sequencing (NGS) of LTRs from 269 HIV-1-infected samples in the Drexel CARES Cohort was used to select gRNAs with predicted broad-spectrum activity. In silico, D-LTR-P4-227913 (package of the top 4 gRNAs) accounted for all detectable genetic variation within the vQS of the 269 samples and the Los Alamos National Laboratory HIV database. In silico secondary structure analyses from NGS indicated extensive TAR stem-loop malformations predicted to inactivate proviral transcription, which was confirmed by reduced viral gene expression in TZM-bl or P4R5 cells. Similarly, a high sensitivity in vitro CRISPR/Cas9 cleavage assay showed that the top-ranked gRNA was the most effective at cleaving patient-derived HIV-1 LTRs from five patients. Furthermore, the D-LTR-P4-227913 was predicted to cleave a median of 96.1% of patient-derived sequences from other HIV subtypes. These results demonstrate that the gRNAs possess broad-spectrum cutting activity and could contribute to an HIV cure.},
}

*CRISPR-Cas Systems
Cohort Studies
Computational Biology
Female
*Gene Editing
Genome, Viral
HIV Infections/*genetics/virology
HIV Long Terminal Repeat/genetics
HIV-1/*genetics/*growth & development/metabolism
Humans
Male
Middle Aged
Proviruses/*genetics
Quasispecies/*genetics
RNA, Guide/*genetics

@article {pmid31729437,
year = {2019},
author = {Datsomor, AK and Olsen, RE and Zic, N and Madaro, A and Bones, AM and Edvardsen, RB and Wargelius, A and Winge, P},
title = {CRISPR/Cas9-mediated editing of Δ5 and Δ6 desaturases impairs Δ8-desaturation and docosahexaenoic acid synthesis in Atlantic salmon (Salmo salar L.).},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {16888},
pmid = {31729437},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Docosahexaenoic Acids/biosynthesis ; Fatty Acid Desaturases/*genetics ; Fatty Acids, Omega-3/biosynthesis ; Fatty Acids, Unsaturated/*biosynthesis ; Gene Editing/*methods ; Lipogenesis/*genetics ; Metabolic Engineering/methods/veterinary ; Mutagenesis/physiology ; Mutation ; *Salmo salar/genetics/growth & development/metabolism ; },
abstract = {The in vivo functions of Atlantic salmon fatty acyl desaturases (fads2), Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2 in long chain polyunsaturated fatty acid (LC-PUFA) synthesis in salmon and fish in general remains to be elucidated. Here, we investigate in vivo functions and in vivo functional redundancy of salmon fads2 using two CRISPR-mediated partial knockout salmon, Δ6abc/5Mt with mutations in Δ6fads2-a, Δ6fads2-b, Δ6fads2-c and Δ5fads2, and Δ6bcMt with mutations in Δ6fads2-b and Δ6fads2-c. F0 fish displaying high degree of gene editing (50-100%) were fed low LC-PUFA and high LC-PUFA diets, the former containing reduced levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids but higher content of linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids, and the latter containing high levels of 20:5n-3 and 22:6n-3 but reduced compositions of 18:2n-6 and 18:3n-3. The Δ6abc/5Mt showed reduced 22:6n-3 levels and accumulated Δ6-desaturation substrates (18:2n-6, 18:3n-3) and Δ5-desaturation substrate (20:4n-3), demonstrating impaired 22:6n-3 synthesis compared to wildtypes (WT). Δ6bcMt showed no effect on Δ6-desaturation compared to WT, suggesting Δ6 Fads2-a as having the predominant Δ6-desaturation activity in salmon, at least in the tissues analyzed. Both Δ6abc/5Mt and Δ6bcMt demonstrated significant accumulation of Δ8-desaturation substrates (20:2n-6, 20:3n-3) when fed low LC-PUFA diet. Additionally, Δ6abc/5Mt demonstrated significant upregulation of the lipogenic transcription regulator, sterol regulatory element binding protein-1 (srebp-1) in liver and pyloric caeca under reduced dietary LC-PUFA. Our data suggest a combined effect of endogenous LC-PUFA synthesis and dietary LC-PUFA levels on srebp-1 expression which ultimately affects LC-PUFA synthesis in salmon. Our data also suggest Δ8-desaturation activities for salmon Δ6 Fads2 enzymes.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems/genetics
Docosahexaenoic Acids/biosynthesis
Fatty Acid Desaturases/*genetics
Fatty Acids, Omega-3/biosynthesis
Fatty Acids, Unsaturated/*biosynthesis
Gene Editing/*methods
Lipogenesis/*genetics
Metabolic Engineering/methods/veterinary
Mutagenesis/physiology
Mutation
*Salmo salar/genetics/growth & development/metabolism

@article {pmid31586114,
year = {2019},
author = {Zhao, X and Wei, W and Pan, H and Nie, J and Chen, D and Zhang, P and Chen, F and Fu, Q and Zuo, E and Lu, Y and Zhang, M},
title = {Identification of the Sex of Pre-implantation Mouse Embryos Using a Marked Y Chromosome and CRISPR/Cas9.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {14315},
doi = {10.1038/s41598-019-50731-x},
pmid = {31586114},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Embryo, Mammalian ; Embryonic Development ; Green Fluorescent Proteins/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic/*embryology/genetics ; *Sex Determination Analysis ; *Y Chromosome ; },
abstract = {Although numerous attempts have been made to alter the sex ratio of the progeny of mammals, the limitations of current technologies have prevented their widespread use in farm animals. The presence or absence of a Y chromosome determines whether a mammalian embryo develops as a male or female, and non-invasive genetic reporters such as fluorescence protein markers have been intensively applied in a variety of fields of research. To develop a non-invasive and instantaneous method for advance determination of the sex of embryos, we developed a Y chromosome-linked eGFP mouse line that stably expresses green fluorescent protein under the control of the CAG promoter. The development of the CRISPR/Cas9 system has made it easy to deliver an exogenous gene to a specific locus of a genome, and linking a tracer to the Y chromosome has simplified the process of predicting the sex of embryos collected by mating a Y-Chr-eGFP transgenic male with a wild-type female. XY embryos appeared green, under a fluorescence microscope, and XX embryos did not. Y chromosome-linked genes were amplified by nested PCR to further confirm the accuracy of this method, and the simultaneous transplantation of green and non-green embryos into foster mothers indicated that 100% accuracy was achieved by this method. Thus, the Y-Chr-eGFP mouse line provides an expeditious and accurate approach for sexing pre-implantation embryos and can be efficiently used for the pre-selection of sex.},
}

Animals
*CRISPR-Cas Systems
Embryo, Mammalian
Embryonic Development
Green Fluorescent Proteins/genetics/metabolism
Mice
Mice, Inbred C57BL
Mice, Transgenic/*embryology/genetics
*Sex Determination Analysis
*Y Chromosome

@article {pmid31506008,
year = {2020},
author = {de Korte, T and Katili, PA and Mohd Yusof, NAN and van Meer, BJ and Saleem, U and Burton, FL and Smith, GL and Clements, P and Mummery, CL and Eschenhagen, T and Hansen, A and Denning, C},
title = {Unlocking Personalized Biomedicine and Drug Discovery with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Fit for Purpose or Forever Elusive?.},
journal = {Annual review of pharmacology and toxicology},
volume = {60},
number = {},
pages = {529-551},
doi = {10.1146/annurev-pharmtox-010919-023309},
pmid = {31506008},
issn = {1545-4304},
support = {RG/15/6/31436/BHF_/British Heart Foundation/United Kingdom ; NC/K000225/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; NC/C013105/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; PG/14/59/31000/BHF_/British Heart Foundation/United Kingdom ; P47352/CRM/BHF_/British Heart Foundation/United Kingdom ; RG/14/1/30588/BHF_/British Heart Foundation/United Kingdom ; NC/C013202/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; SP/15/9/31605/BHF_/British Heart Foundation/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cardiotoxicity/*prevention & control ; Drug Development/methods ; Drug Discovery/*methods ; Genetic Engineering ; Humans ; Induced Pluripotent Stem Cells/cytology ; Myocytes, Cardiac/cytology/*drug effects ; Precision Medicine/methods ; },
abstract = {In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease.},
}

Animals
CRISPR-Cas Systems/genetics
Cardiotoxicity/*prevention & control
Drug Development/methods
Drug Discovery/*methods
Genetic Engineering
Humans
Induced Pluripotent Stem Cells/cytology
Myocytes, Cardiac/cytology/*drug effects
Precision Medicine/methods

@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 {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.

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.

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.

CONCLUSION: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release.},
}

@article {pmid33159392,
year = {2020},
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 = {},
number = {},
pages = {},
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},
doi = {10.1038/s42003-020-01366-6},
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},
doi = {10.1038/s41467-020-19415-3},
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 ; },
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 & differentiation},
volume = {},
number = {},
pages = {},
doi = {10.1111/dgd.12700},
pmid = {33155277},
issn = {1440-169X},
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 to obtain 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},
doi = {10.1038/s41598-020-76193-0},
pmid = {33154469},
issn = {2045-2322},
support = {EXS2026//DFG excellence cluster Cardiopulmonary Institute (CPI)/ ; TPA01//SFB1039/ ; Transregio TRR267, TPA06//Deutsche Forschungsgemeinschaft/ ; Transregio TRR267 TPA04//Deutsche Forschungsgemeinschaft/ ; },
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 = {},
doi = {10.3390/microorganisms8111720},
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 {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.},
}

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

@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.},
}

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

@article {pmid32821942,
year = {2020},
author = {Veeneman, B and Gao, Y and Grant, J and Fruhling, D and Ahn, J and Bosbach, B and Bienkowska, J and Follettie, M and Arndt, K and Myers, J and Zhong, W},
title = {PINCER: improved CRISPR/Cas9 screening by efficient cleavage at conserved residues.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9462-9477},
pmid = {32821942},
issn = {1362-4962},
mesh = {*Algorithms ; Amino Acid Sequence ; Amino Acids/genetics ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Conserved Sequence ; *Databases, Genetic ; Enzymes/genetics/metabolism ; Genome ; Genomic Library ; Humans ; Mice ; Proteins/*genetics/*metabolism ; RNA, Guide/genetics ; Reproducibility of Results ; Thymidine/genetics ; },
abstract = {CRISPR/Cas9 functional genomic screens have emerged as essential tools in drug target discovery. However, the sensitivity of available genome-wide CRISPR libraries is impaired by guides which inefficiently abrogate gene function. While Cas9 cleavage efficiency optimization and essential domain targeting have been developed as independent guide design rationales, no library has yet combined these into a single cohesive strategy to knock out gene function. Here, in a massive reanalysis of CRISPR tiling data using the most comprehensive feature database assembled, we determine which features of guides and their targets best predict activity and how to best combine them into a single guide design algorithm. We present the ProteIN ConsERvation (PINCER) genome-wide CRISPR library, which for the first time combines enzymatic efficiency optimization with conserved length protein region targeting, and also incorporates domains, coding sequence position, U6 termination (TTT), restriction sites, polymorphisms and specificity. Finally, we demonstrate superior performance of the PINCER library compared to alternative genome-wide CRISPR libraries in head-to-head validation. PINCER is available for individual gene knockout and genome-wide screening for both the human and mouse genomes.},
}

*Algorithms
Amino Acid Sequence
Amino Acids/genetics
Animals
*CRISPR-Cas Systems
Cell Line
Conserved Sequence
*Databases, Genetic
Enzymes/genetics/metabolism
Genome
Genomic Library
Humans
Mice
Proteins/*genetics/*metabolism
RNA, Guide/genetics
Reproducibility of Results
Thymidine/genetics

@article {pmid32810208,
year = {2020},
author = {Carullo, NVN and Phillips Iii, RA and Simon, RC and Soto, SAR and Hinds, JE and Salisbury, AJ and Revanna, JS and Bunner, KD and Ianov, L and Sultan, FA and Savell, KE and Gersbach, CA and Day, JJ},
title = {Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9550-9570},
pmid = {32810208},
issn = {1362-4962},
support = {R01 MH114990/MH/NIMH NIH HHS/United States ; DP1 DA039650/DA/NIDA NIH HHS/United States ; K99 DA034681/DA/NIDA NIH HHS/United States ; R21 DA041878/DA/NIDA NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; CREB-Binding Protein/genetics/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Chromatin/metabolism ; *Enhancer Elements, Genetic ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; Neurons/cytology/*physiology ; Proto-Oncogene Proteins c-fos/genetics ; RNA/*physiology ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering ; Rats ; Reproducibility of Results ; Sequence Analysis, RNA ; Single Molecule Imaging ; },
abstract = {Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.},
}

Animals
CREB-Binding Protein/genetics/metabolism
CRISPR-Cas Systems
Cells, Cultured
Chromatin/metabolism
*Enhancer Elements, Genetic
*Gene Expression Regulation
HEK293 Cells
Humans
Neurons/cytology/*physiology
Proto-Oncogene Proteins c-fos/genetics
RNA/*physiology
RNA, Messenger/genetics/metabolism
RNA, Small Interfering
Rats
Reproducibility of Results
Sequence Analysis, RNA
Single Molecule Imaging

@article {pmid32797168,
year = {2020},
author = {Chien, JC and Tabet, E and Pinkham, K and da Hora, CC and Chang, JC and Lin, S and Badr, CE and Lai, CP},
title = {A multiplexed bioluminescent reporter for sensitive and non-invasive tracking of DNA double strand break repair dynamics in vitro and in vivo.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {e100},
pmid = {32797168},
issn = {1362-4962},
support = {K22 CA197053/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Copepoda/enzymology ; DNA End-Joining Repair ; Female ; *Genes, Reporter ; HEK293 Cells ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Luciferases/*genetics/metabolism ; Mice ; Mice, Nude ; Multiplex Polymerase Chain Reaction/methods ; Optical Imaging/methods ; Rad51 Recombinase/genetics/metabolism ; *Recombinational DNA Repair ; Sequence Analysis, DNA/methods ; },
abstract = {Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1-10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.},
}

Animals
CRISPR-Cas Systems
Cell Line, Tumor
Copepoda/enzymology
DNA End-Joining Repair
Female
*Genes, Reporter
HEK293 Cells
High-Throughput Nucleotide Sequencing/methods
Humans
Luciferases/*genetics/metabolism
Mice
Mice, Nude
Multiplex Polymerase Chain Reaction/methods
Optical Imaging/methods
Rad51 Recombinase/genetics/metabolism
*Recombinational DNA Repair
Sequence Analysis, DNA/methods

@article {pmid32797156,
year = {2020},
author = {Iwasaki, RS and Batey, RT},
title = {SPRINT: a Cas13a-based platform for detection of small molecules.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {e101},
pmid = {32797156},
issn = {1362-4962},
support = {R01 GM073850/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/genetics/*metabolism ; Biosensing Techniques/*methods ; CRISPR-Associated Proteins/genetics/*metabolism ; CRISPR-Cas Systems ; Endodeoxyribonucleases/genetics/*metabolism ; Enzyme Assays/*methods ; Fluorescent Dyes/chemistry ; Leptotrichia ; Ligands ; Nucleic Acid Synthesis Inhibitors/*analysis/pharmacology ; Riboswitch ; Rifampin/analysis ; Transcription Factors/metabolism ; Transcription, Genetic/drug effects ; },
abstract = {Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.},
}

Bacterial Proteins/genetics/*metabolism
Biosensing Techniques/*methods
CRISPR-Associated Proteins/genetics/*metabolism
CRISPR-Cas Systems
Endodeoxyribonucleases/genetics/*metabolism
Enzyme Assays/*methods
Fluorescent Dyes/chemistry
Leptotrichia
Ligands
Nucleic Acid Synthesis Inhibitors/*analysis/pharmacology
Riboswitch
Rifampin/analysis
Transcription Factors/metabolism
Transcription, Genetic/drug effects

@article {pmid32766783,
year = {2020},
author = {Steinberger, J and Shen, L and J Kiniry, S and Naineni, SK and Cencic, R and Amiri, M and Aboushawareb, SAE and Chu, J and Maïga, RI and Yachnin, BJ and Robert, F and Sonenberg, N and Baranov, PV and Pelletier, J},
title = {Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5' leader regions.},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9521-9537},
pmid = {32766783},
issn = {1362-4962},
support = {210692/Z/18//WT_/Wellcome Trust/United Kingdom ; FDN-148366//CIHR/Canada ; },
mesh = {*5' Untranslated Regions ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Resistance, Neoplasm/drug effects/*genetics ; Eukaryotic Initiation Factor-4A/antagonists & inhibitors/*genetics/metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy/genetics/pathology ; Mutation ; Ribosomes/genetics/metabolism ; Sterols/*pharmacology ; },
abstract = {Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5' leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.},
}

*5' Untranslated Regions
CRISPR-Cas Systems
Cell Line, Tumor
Drug Resistance, Neoplasm/drug effects/*genetics
Eukaryotic Initiation Factor-4A/antagonists & inhibitors/*genetics/metabolism
Humans
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy/genetics/pathology
Mutation
Ribosomes/genetics/metabolism
Sterols/*pharmacology

@article {pmid32694105,
year = {2020},
author = {Chen, YN and Lu, J},
title = {[Application of CRISPR/Cas9 mediated gene editing in trees].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {7},
pages = {657-668},
doi = {10.16288/j.yczz.20-092},
pmid = {32694105},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Plants/genetics ; *Trees/genetics ; },
abstract = {The CRISPR/Cas9 system, which can induce precise modifications at a target gene, has been recognized as the most promising gene editing technology, and has played an important role in precision crop breeding. It also provides a new strategy for fundamental researches and molecular breeding of forest trees. Recently, CRISPR/Cas9-mediated gene editing has been applied more extensively in tree genetic studies. It has not only succeeded in developing new drought- resistance or disease-resistant cultivars, but also shows a great potential in regulating lignin biosynthesis and shortening the breeding cycle of forest trees. In this review, we summarize the application and advance of CRISPR/Cas9 in gene function identification and genetic improvement of forest plants. We also discuss the related problems and future perspectives. This review aims to provide a useful reference for tree functional genomics and genetic engineering breeding.},
}

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

@article {pmid32694104,
year = {2020},
author = {Li, GL and Yang, SX and Wu, ZF and Zhang, XW},
title = {[Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {7},
pages = {641-656},
doi = {10.16288/j.yczz.20-056},
pmid = {32694104},
issn = {0253-9772},
mesh = {Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Knock-In Techniques ; Humans ; Recombinational DNA Repair ; },
abstract = {Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.},
}

Animals
CRISPR-Associated Protein 9
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Gene Knock-In Techniques
Humans
Recombinational DNA Repair

@article {pmid32672817,
year = {2020},
author = {Tian, J and Yang, G and Gu, Y and Sun, X and Lu, Y and Jiang, W},
title = {Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces.},
journal = {Nucleic acids research},
volume = {48},
number = {14},
pages = {8188-8202},
pmid = {32672817},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; Industrial Microbiology/*methods ; *Quorum Sensing ; Sirolimus/metabolism ; Streptomyces/*genetics/metabolism ; },
abstract = {Quorum-sensing (QS) mediated dynamic regulation has emerged as an effective strategy for optimizing product titers in microbes. However, these QS-based circuits are often created on heterologous systems and require careful tuning via a tedious testing/optimization process. This hampers their application in industrial microbes. Here, we design a novel QS circuit by directly integrating an endogenous QS system with CRISPRi (named EQCi) in the industrial rapamycin-producing strain Streptomyces rapamycinicus. EQCi combines the advantages of both the QS system and CRISPRi to enable tunable, autonomous, and dynamic regulation of multiple targets simultaneously. Using EQCi, we separately downregulate three key nodes in essential pathways to divert metabolic flux towards rapamycin biosynthesis and significantly increase its titers. Further application of EQCi to simultaneously regulate these three key nodes with fine-tuned repression strength boosts the rapamycin titer by ∼660%, achieving the highest reported titer (1836 ± 191 mg/l). Notably, compared to static engineering strategies, which result in growth arrest and suboptimal rapamycin titers, EQCi-based regulation substantially promotes rapamycin titers without affecting cell growth, indicating that it can achieve a trade-off between essential pathways and product synthesis. Collectively, this study provides a convenient and effective strategy for strain improvement and shows potential for application in other industrial microorganisms.},
}

*CRISPR-Cas Systems
*Gene Expression Regulation, Bacterial
Industrial Microbiology/*methods
*Quorum Sensing
Sirolimus/metabolism
Streptomyces/*genetics/metabolism

@article {pmid32632303,
year = {2020},
author = {Song, M and Kim, HK and Lee, S and Kim, Y and Seo, SY and Park, J and Choi, JW and Jang, H and Shin, JH and Min, S and Quan, Z and Kim, JH and Kang, HC and Yoon, S and Kim, HH},
title = {Sequence-specific prediction of the efficiencies of adenine and cytosine base editors.},
journal = {Nature biotechnology},
volume = {38},
number = {9},
pages = {1037-1043},
doi = {10.1038/s41587-020-0573-5},
pmid = {32632303},
issn = {1546-1696},
mesh = {*Adenine ; Aminohydrolases/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; *Cytosine ; Cytosine Deaminase/metabolism ; Gene Editing/*methods ; Genetic Engineering ; Genome, Human/genetics ; HEK293 Cells ; Humans ; Point Mutation ; RNA, Guide/genetics ; Targeted Gene Repair/*methods ; },
abstract = {Base editors, including adenine base editors (ABEs)1 and cytosine base editors (CBEs)2,3, are widely used to induce point mutations. However, determining whether a specific nucleotide in its genomic context can be edited requires time-consuming experiments. Furthermore, when the editable window contains multiple target nucleotides, various genotypic products can be generated. To develop computational tools to predict base-editing efficiency and outcome product frequencies, we first evaluated the efficiencies of an ABE and a CBE and the outcome product frequencies at 13,504 and 14,157 target sequences, respectively, in human cells. We found that there were only modest asymmetric correlations between the activities of the base editors and Cas9 at the same targets. Using deep-learning-based computational modeling, we built tools to predict the efficiencies and outcome frequencies of ABE- and CBE-directed editing at any target sequence, with Pearson correlations ranging from 0.50 to 0.95. These tools and results will facilitate modeling and therapeutic correction of genetic diseases by base editing.},
}

*Adenine
Aminohydrolases/metabolism
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems
*Cytosine
Cytosine Deaminase/metabolism
Gene Editing/*methods
Genetic Engineering
Genome, Human/genetics
HEK293 Cells
Humans
Point Mutation
RNA, Guide/genetics
Targeted Gene Repair/*methods

@article {pmid32619241,
year = {2020},
author = {Indikova, I and Indik, S},
title = {Highly efficient 'hit-and-run' genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts.},
journal = {Nucleic acids research},
volume = {48},
number = {14},
pages = {8178-8187},
pmid = {32619241},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Vectors/genetics ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Nanoparticles/chemistry ; *Response Elements ; THP-1 Cells ; Transduction, Genetic/methods ; },
abstract = {The application of gene-editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and gag/pol gene expression (the presence of Rev responsive element, RRE) greatly enhanced the encapsidation of the fusion protein and resulted in the production of highly efficient lentivector nanoparticles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors led to >98% disruption of the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, dose-controlled and tissue-specific delivery of genome editing enzymes to cells and it may be suitable for simultaneous endogenous gene disruption and a transgene delivery.},
}

CRISPR-Associated Protein 9/genetics/*metabolism
*CRISPR-Cas Systems
Gene Editing/*methods
Genetic Vectors/genetics
Green Fluorescent Proteins/genetics/metabolism
HEK293 Cells
Humans
Lentivirus/genetics
Nanoparticles/chemistry
*Response Elements
THP-1 Cells
Transduction, Genetic/methods

@article {pmid32572256,
year = {2020},
author = {Gretarsson, KH and Hackett, JA},
title = {Dppa2 and Dppa4 counteract de novo methylation to establish a permissive epigenome for development.},
journal = {Nature structural & molecular biology},
volume = {27},
number = {8},
pages = {706-716},
doi = {10.1038/s41594-020-0445-1},
pmid = {32572256},
issn = {1545-9985},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *DNA Methylation ; Epigenome ; Gene Expression Regulation, Developmental ; Long Interspersed Nucleotide Elements ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Nuclear Proteins/*genetics/metabolism ; Single-Cell Analysis ; Transcription Factors/*genetics/metabolism ; },
abstract = {Early mammalian development entails genome-wide epigenome remodeling, including DNA methylation erasure and reacquisition, which facilitates developmental competence. To uncover the mechanisms that orchestrate DNA methylation dynamics, we coupled a single-cell ratiometric DNA methylation reporter with unbiased CRISPR screening in murine embryonic stem cells (ESCs). We identify key genes and regulatory pathways that drive global DNA hypomethylation, and characterize roles for Cop1 and Dusp6. We also identify Dppa2 and Dppa4 as essential safeguards of focal epigenetic states. In their absence, developmental genes and evolutionarily young LINE1 elements, which are specifically bound by DPPA2, lose H3K4me3 and gain ectopic de novo DNA methylation in pluripotent cells. Consequently, lineage-associated genes and LINE1 acquire a repressive epigenetic memory, which renders them incompetent for activation during future lineage specification. Dppa2/4 thereby sculpt the pluripotent epigenome by facilitating H3K4me3 and bivalency to counteract de novo methylation, a function co-opted by evolutionarily young LINE1 to evade epigenetic decommissioning.},
}

Animals
CRISPR-Cas Systems
Cell Line
*DNA Methylation
Epigenome
Gene Expression Regulation, Developmental
Long Interspersed Nucleotide Elements
Mice
Mouse Embryonic Stem Cells/cytology/*metabolism
Nuclear Proteins/*genetics/metabolism
Single-Cell Analysis
Transcription Factors/*genetics/metabolism

@article {pmid32513585,
year = {2020},
author = {Marzec, M and Hensel, G},
title = {Prime Editing: Game Changer for Modifying Plant Genomes.},
journal = {Trends in plant science},
volume = {25},
number = {8},
pages = {722-724},
doi = {10.1016/j.tplants.2020.05.008},
pmid = {32513585},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems/genetics ; DNA ; *Gene Editing ; Genome, Plant/genetics ; Humans ; Mutation ; },
abstract = {Prime editing, developed by Anzalone et al., brings genome editing to a new level, because this approach allows introduction of all mutation types, including insertions, deletions, and all putative 12 types of base-to-base conversions. Previously tested in human cells, this technique has been adapted for use in plants by Lin et al.},
}

*CRISPR-Cas Systems/genetics
DNA
*Gene Editing
Genome, Plant/genetics
Humans
Mutation

@article {pmid32402659,
year = {2020},
author = {Vangheluwe, N and Swinnen, G and de Koning, R and Meyer, P and Houben, M and Huybrechts, M and Sajeev, N and Rienstra, J and Boer, D},
title = {Give CRISPR a Chance: the GeneSprout Initiative.},
journal = {Trends in plant science},
volume = {25},
number = {7},
pages = {624-627},
doi = {10.1016/j.tplants.2020.04.011},
pmid = {32402659},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Plant Breeding ; Plants/genetics ; },
abstract = {Did you know that a group of early-career researchers launched an initiative enabling open dialog on new plant breeding techniques, such as genome editing? We developed a wide-ranging initiative that aims to facilitate public engagement and provide a platform for young plant scientists to encourage participation in science communication.},
}

*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Plant Breeding
Plants/genetics

@article {pmid32393821,
year = {2020},
author = {Simoni, A and Hammond, AM and Beaghton, AK and Galizi, R and Taxiarchi, C and Kyrou, K and Meacci, D and Gribble, M and Morselli, G and Burt, A and Nolan, T and Crisanti, A},
title = {A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae.},
journal = {Nature biotechnology},
volume = {38},
number = {9},
pages = {1054-1060},
doi = {10.1038/s41587-020-0508-1},
pmid = {32393821},
issn = {1546-1696},
mesh = {Animals ; Anopheles/*genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/genetics/metabolism ; Female ; Gene Drive Technology/*methods ; Insect Proteins/genetics/metabolism ; Malaria/prevention & control/*transmission ; Male ; Mosquito Control ; Mosquito Vectors/*genetics ; Sex Determination Processes/*genetics ; X Chromosome/genetics/metabolism ; },
abstract = {Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10-14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control.},
}

Animals
Anopheles/*genetics
CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems/genetics
Endodeoxyribonucleases/genetics/metabolism
Female
Gene Drive Technology/*methods
Insect Proteins/genetics/metabolism
Malaria/prevention & control/*transmission
Male
Mosquito Control
Mosquito Vectors/*genetics
Sex Determination Processes/*genetics
X Chromosome/genetics/metabolism

@article {pmid32376086,
year = {2020},
author = {Zheng, HX and Sun, X and Zhang, XS and Sui, N},
title = {m6A Editing: New Tool to Improve Crop Quality?.},
journal = {Trends in plant science},
volume = {25},
number = {9},
pages = {859-867},
doi = {10.1016/j.tplants.2020.04.005},
pmid = {32376086},
issn = {1878-4372},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Plant Development ; Plants/genetics ; RNA ; },
abstract = {N6-methyladenosine (m6A) is the most common type of eukaryotic mRNA modification. It plays an important role in regulating plant growth and development and stress resistance. m6A modification influences nearly all aspects of RNA metabolism and functionality and has great potential for improving crop quality. However, changing m6A modification levels as a whole may have unpredictable effects, making it impossible to accurately predict the effect of specific m6A modifications on RNA. In this opinion article, the main challenges and possible solutions for exploring m6A modification functions in plant systems are discussed. An m6A editing platform that uses new high-throughput methods to identify m6A modification at single-base resolution, and genome editing for selective editing of specific m6A sites for crop improvement is proposed.},
}

*CRISPR-Cas Systems
*Gene Editing
Plant Development
Plants/genetics
RNA

@article {pmid32372479,
year = {2020},
author = {Hua, K and Jiang, Y and Tao, X and Zhu, JK},
title = {Precision genome engineering in rice using prime editing system.},
journal = {Plant biotechnology journal},
volume = {18},
number = {11},
pages = {2167-2169},
pmid = {32372479},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/genetics ; Gene Editing ; *Oryza/genetics ; },
}

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