@article {pmid30354961,
year = {2018},
author = {Pasricha, SR and Drakesmith, H},
title = {Hemoglobinopathies in the Fetal Position.},
journal = {The New England journal of medicine},
volume = {379},
number = {17},
pages = {1675-1677},
doi = {10.1056/NEJMcibr1809628},
pmid = {30354961},
issn = {1533-4406},
mesh = {Anemia, Sickle Cell/*drug therapy/genetics ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Erythroid Cells/metabolism ; Fetal Hemoglobin/*genetics ; *Gene Expression Regulation ; Humans ; Molecular Targeted Therapy ; eIF-2 Kinase/*antagonists & inhibitors/genetics/metabolism ; },
}

Anemia, Sickle Cell/*drug therapy/genetics
CRISPR-Cas Systems
Carrier Proteins/genetics
Erythroid Cells/metabolism
Fetal Hemoglobin/*genetics
*Gene Expression Regulation
Humans
Molecular Targeted Therapy
eIF-2 Kinase/*antagonists & inhibitors/genetics/metabolism

@article {pmid30151606,
year = {2018},
author = {Dong, Z and Huang, L and Dong, F and Hu, Z and Qin, Q and Long, J and Cao, M and Chen, P and Lu, C and Pan, MH},
title = {Establishment of a baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms.},
journal = {Applied microbiology and biotechnology},
volume = {102},
number = {21},
pages = {9255-9265},
doi = {10.1007/s00253-018-9295-8},
pmid = {30151606},
issn = {1432-0614},
support = {31472153//National Natural Science Foundation of China/ ; 31572466//National Natural Science Foundation of China/ ; CARS-18//China Agriculture Research System/ ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; Baculoviridae/*genetics ; Bombyx/*genetics/virology ; CRISPR-Cas Systems/*genetics ; Gene Targeting/methods ; Genetic Vectors/genetics ; Nucleopolyhedrovirus/genetics ; Pest Control/methods ; Promoter Regions, Genetic/genetics ; Transcription, Genetic/genetics ; },
abstract = {The CRISPR/Cas9 system is a powerful genetic engineering technique that has been widely used in gene therapy, as well as in the development of novel antimicrobials and transgenic insects. However, several challenges, including the lack of effective host target genes and the off-target effects, limit the application of CRISPR/Cas9 in insects. To mitigate these difficulties, we established a highly efficient virus-inducible CRISPR/Cas9 system in transgenic silkworms. This system includes the baculovirus-inducible promoter 39K, which directs transcription of the gene encoding, the Cas9 protein, and the U6 promoter which targets the sgATAD3A site of the ATPase family AAA domain-containing protein 3 (ATAD3A) gene. The double-positive transgenic line sgATAD3A×39K-Cas9 (ATAD3A-KO) was obtained by hybridization; antiviral activity in this hybrid transgenic line is induced only after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The BmNPV-inducible system significantly reduced off-target effects and did not affect the economically important characteristics of the transgenic silkworms. Most importantly, this novel system efficiently and consistently edited target genes, inhibiting BmNPV replication after the transgenic silkworms were inoculated with occlusion bodies (OBs). The suppression of BmNPV by the virus-inducible system was comparable to that of the stably expressed CRISPR/Cas9 system. Therefore, we successfully established a highly efficient BmNPV-inducible ATAD3A-KO transgenic silkworm line, with improved gene targeting specificity and antiviral efficiency. Our study thereby provides insights into the treatment of infectious diseases and into the control of insect pests.},
}

Animals
Animals, Genetically Modified/*genetics
Baculoviridae/*genetics
Bombyx/*genetics/virology
CRISPR-Cas Systems/*genetics
Gene Targeting/methods
Genetic Vectors/genetics
Nucleopolyhedrovirus/genetics
Pest Control/methods
Promoter Regions, Genetic/genetics
Transcription, Genetic/genetics

@article {pmid29604353,
year = {2018},
author = {Wang, S and Hong, W and Dong, S and Zhang, ZT and Zhang, J and Wang, L and Wang, Y},
title = {Genome engineering of Clostridium difficile using the CRISPR-Cas9 system.},
journal = {Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases},
volume = {24},
number = {10},
pages = {1095-1099},
doi = {10.1016/j.cmi.2018.03.026},
pmid = {29604353},
issn = {1469-0691},
mesh = {Animals ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clostridium difficile/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Deletion ; Genetic Engineering/methods ; Genome, Bacterial/genetics ; Humans ; Plasmids/genetics ; },
abstract = {OBJECTIVES: Clostridium difficile is a notorious pathogenic species that can cause severe gastrointestinal infections in humans and animals. C. difficile infection (CDI) results in thousands of deaths worldwide every year. The elucidation of related mechanisms of CDI and exploration of potential therapeutic strategies are largely delayed due to the lack of efficient genetic engineering tools for C. difficile strains.

METHODS: Plasmids carrying the CRISPR-Cas9 system were constructed and transformed into C. difficile through conjugation. Mutants were identified using colony PCR with primers annealing to the regions flanking the target gene deletion/integration locus. Heat-survival assay was used to compare the sporulation frequency between the mutant with spo0A deletion and the wild type strain. The fluorescence in the mutant with the insertion of the green fluorescent protein (GFP) gene was inspected under a fluorescent microscope.

RESULTS: An efficient genome editing tool was developed for C. difficile based on the CRISPR-Cas9 system. With this tool, spo0A was deleted with a 100% mutation efficiency. Conversely, an anaerobic GFP gene was successfully inserted into the C. difficile chromosome (with a mutation efficiency of 80%).

CONCLUSIONS: The developed CRISPR-Cas9-based genome engineering tool will facilitate functional genomic studies in C. difficile as well as the elucidation of mechanisms related to host-bacteria interaction and pathogenesis of CDI. This will be highly beneficial for the development of innovative strategies for CDI diagnostics and therapies.},
}

Animals
Bacterial Proteins/genetics
CRISPR-Cas Systems/*genetics
Clostridium difficile/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Deletion
Genetic Engineering/methods
Genome, Bacterial/genetics
Humans
Plasmids/genetics

@article {pmid29540148,
year = {2018},
author = {Vazquez, N and Sanchez, L and Marks, R and Martinez, E and Fanniel, V and Lopez, A and Salinas, A and Flores, I and Hirschmann, J and Gilkerson, R and Schuenzel, E and Dearth, R and Halaby, R and Innis-Whitehouse, W and Keniry, M},
title = {A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.},
journal = {BMC molecular biology},
volume = {19},
number = {1},
pages = {3},
doi = {10.1186/s12867-018-0105-8},
pmid = {29540148},
issn = {1471-2199},
support = {UTRGV College of Sciences (COS) Seed Grant//UTRGV COS/International ; NIH 1SC3GM11666901/GM/NIGMS NIH HHS/United States ; USDA H.S.I. 2016-38422-25760//National Institute of Food and Agriculture/International ; 1463991//National Science Foundation/International ; NIH 5R25GM10086606/GM/NIGMS NIH HHS/United States ; USDA Step 2 2015-38422-24061//National Institute of Food and Agriculture/International ; NSF 1463991//National Science Foundation/International ; NSF Advance 1209210//National Science Foundation/International ; HHMI 52007568/HHMI/Howard Hughes Medical Institute/United States ; SC3 GM116669/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Deoxyribonuclease I/metabolism ; Forkhead Box Protein O3/*genetics ; Gene Editing/*methods ; Genetic Vectors ; HEK293 Cells ; Homologous Recombination ; Humans ; Male ; Mutation ; Plasmids/*genetics ; RNA, Guide/metabolism ; },
abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.

RESULTS: We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.

CONCLUSIONS: Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).},
}

*CRISPR-Cas Systems
Cell Line
Deoxyribonuclease I/metabolism
Forkhead Box Protein O3/*genetics
Gene Editing/*methods
Genetic Vectors
HEK293 Cells
Homologous Recombination
Humans
Male
Mutation
Plasmids/*genetics
RNA, Guide/metabolism

@article {pmid28917642,
year = {2018},
author = {Timin, AS and Muslimov, AR and Lepik, KV and Epifanovskaya, OS and Shakirova, AI and Mock, U and Riecken, K and Okilova, MV and Sergeev, VS and Afanasyev, BV and Fehse, B and Sukhorukov, GB},
title = {Efficient gene editing via non-viral delivery of CRISPR-Cas9 system using polymeric and hybrid microcarriers.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {14},
number = {1},
pages = {97-108},
doi = {10.1016/j.nano.2017.09.001},
pmid = {28917642},
issn = {1549-9642},
mesh = {*CRISPR-Cas Systems ; Drug Carriers ; Fluorescence ; *Gene Editing ; Gene Transfer Techniques ; Green Fluorescent Proteins/antagonists & inhibitors/genetics/metabolism ; HEK293 Cells ; Humans ; Lycopersicon esculentum/genetics/*metabolism ; Polymers/*chemistry ; Silicon Dioxide/chemistry ; },
abstract = {CRISPR-Cas9 is a revolutionary genome-editing technology that has enormous potential for the treatment of genetic diseases. However, the lack of efficient and safe, non-viral delivery systems has hindered its clinical application. Here, we report on the application of polymeric and hybrid microcarriers, made of degradable polymers such as polypeptides and polysaccharides and modified by silica shell, for delivery of all CRISPR-Cas9 components. We found that these microcarriers mediate more efficient transfection than a commercially available liposome-based transfection reagent (>70% vs. <50% for mRNA, >40% vs. 20% for plasmid DNA). For proof-of-concept, we delivered CRISPR-Cas9 components using our capsules to dTomato-expressing HEK293T cells-a model, in which loss of red fluorescence indicates successful gene editing. Notably, transfection of indicator cells translated in high-level dTomato knockout in approx. 70% of transfected cells. In conclusion, we have provided proof-of-principle that our micro-sized containers represent promising non-viral platforms for efficient and safe gene editing.},
}

*CRISPR-Cas Systems
Drug Carriers
Fluorescence
*Gene Editing
Gene Transfer Techniques
Green Fluorescent Proteins/antagonists & inhibitors/genetics/metabolism
HEK293 Cells
Humans
Lycopersicon esculentum/genetics/*metabolism
Polymers/*chemistry
Silicon Dioxide/chemistry

@article {pmid28803899,
year = {2017},
author = {Yang, N and Wang, R and Zhao, Y},
title = {Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology.},
journal = {Molecular plant},
volume = {10},
number = {9},
pages = {1141-1143},
doi = {10.1016/j.molp.2017.08.001},
pmid = {28803899},
issn = {1752-9867},
support = {R01 GM114660/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics/growth & development ; Gene Editing/*methods ; *Genomics ; },
}

CRISPR-Cas Systems/*genetics
Crops, Agricultural/*genetics/growth & development
Gene Editing/*methods
*Genomics

@article {pmid28645639,
year = {2017},
author = {Meng, X and Yu, H and Zhang, Y and Zhuang, F and Song, X and Gao, S and Gao, C and Li, J},
title = {Construction of a Genome-Wide Mutant Library in Rice Using CRISPR/Cas9.},
journal = {Molecular plant},
volume = {10},
number = {9},
pages = {1238-1241},
doi = {10.1016/j.molp.2017.06.006},
pmid = {28645639},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Library ; *Genomics ; *Mutation ; Oryza/*genetics ; },
}

CRISPR-Cas Systems/*genetics
*Gene Library
*Genomics
*Mutation
Oryza/*genetics

@article {pmid28645638,
year = {2017},
author = {Lu, Y and Ye, X and Guo, R and Huang, J and Wang, W and Tang, J and Tan, L and Zhu, JK and Chu, C and Qian, Y},
title = {Genome-wide Targeted Mutagenesis in Rice Using the CRISPR/Cas9 System.},
journal = {Molecular plant},
volume = {10},
number = {9},
pages = {1242-1245},
doi = {10.1016/j.molp.2017.06.007},
pmid = {28645638},
issn = {1752-9867},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Genome, Plant/*genetics ; Mutagenesis/*genetics ; },
}

Base Sequence
CRISPR-Cas Systems/*genetics
Genome, Plant/*genetics
Mutagenesis/*genetics

@article {pmid28315751,
year = {2017},
author = {Wang, M and Lu, Y and Botella, JR and Mao, Y and Hua, K and Zhu, JK},
title = {Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System.},
journal = {Molecular plant},
volume = {10},
number = {7},
pages = {1007-1010},
doi = {10.1016/j.molp.2017.03.002},
pmid = {28315751},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Repair/*genetics ; Geminiviridae/genetics/*physiology ; Gene Targeting/*methods ; Oryza/*genetics ; *Sequence Homology, Nucleic Acid ; },
}

CRISPR-Cas Systems/*genetics
DNA Repair/*genetics
Geminiviridae/genetics/*physiology
Gene Targeting/*methods
Oryza/*genetics
*Sequence Homology, Nucleic Acid

@article {pmid28272508,
year = {2017},
author = {Mittal, K and Choi, DH and Ogden, A and Donthamsetty, S and Melton, BD and Gupta, MV and Pannu, V and Cantuaria, G and Varambally, S and Reid, MD and Jonsdottir, K and Janssen, EA and Aleskandarany, MA and Ellis, IO and Rakha, EA and Rida, PC and Aneja, R},
title = {Amplified centrosomes and mitotic index display poor concordance between patient tumors and cultured cancer cells.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {43984},
doi = {10.1038/srep43984},
pmid = {28272508},
issn = {2045-2322},
support = {U01 CA179671/CA/NCI NIH HHS/United States ; R01 CA169127/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast Neoplasms/metabolism/*pathology ; CRISPR-Cas Systems/genetics ; Cell Hypoxia ; Cell Line, Tumor ; Centrosome/*metabolism ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Male ; Mice ; Mice, Nude ; Microscopy, Fluorescence ; Mitotic Index ; Pancreatic Neoplasms/metabolism/*pathology ; Transplantation, Heterologous ; Urinary Bladder Neoplasms/metabolism/pathology ; },
abstract = {Centrosome aberrations (CA) and abnormal mitoses are considered beacons of malignancy. Cancer cell doubling times in patient tumors are longer than in cultures, but differences in CA between tumors and cultured cells are uncharacterized. We compare mitoses and CA in patient tumors, xenografts, and tumor cell lines. We find that mitoses are rare in patient tumors compared with xenografts and cell lines. Contrastingly, CA is more extensive in patient tumors and xenografts (~35-50% cells) than cell lines (~5-15%), although CA declines in patient-derived tumor cells over time. Intratumoral hypoxia may explain elevated CA in vivo because exposure of cultured cells to hypoxia or mimicking hypoxia pharmacologically or genetically increases CA, and HIF-1α and hypoxic gene signature expression correlate with CA and centrosomal gene signature expression in breast tumors. These results highlight the importance of utilizing low-passage-number patient-derived cell lines in studying CA to more faithfully recapitulate in vivo cellular phenotypes.},
}

Animals
Breast Neoplasms/metabolism/*pathology
CRISPR-Cas Systems/genetics
Cell Hypoxia
Cell Line, Tumor
Centrosome/*metabolism
Female
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism
Male
Mice
Mice, Nude
Microscopy, Fluorescence
Mitotic Index
Pancreatic Neoplasms/metabolism/*pathology
Transplantation, Heterologous
Urinary Bladder Neoplasms/metabolism/pathology

@article {pmid28195574,
year = {2017},
author = {Bengtsson, NE and Hall, JK and Odom, GL and Phelps, MP and Andrus, CR and Hawkins, RD and Hauschka, SD and Chamberlain, JR and Chamberlain, JS},
title = {Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14454},
doi = {10.1038/ncomms14454},
pmid = {28195574},
issn = {2041-1723},
support = {R01 AR044533/AR/NIAMS NIH HHS/United States ; U54 AR065139/AR/NIAMS NIH HHS/United States ; U54 HD083091/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dependovirus/genetics ; Disease Models, Animal ; Dystrophin/*genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors ; High-Throughput Nucleotide Sequencing ; Male ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/physiopathology ; Muscular Dystrophy, Duchenne/*genetics/*metabolism/*physiopathology/therapy ; Mutation ; Myocardium ; Neuromuscular Diseases/therapy ; RNA, Guide ; Sequence Deletion ; },
abstract = {Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.},
}

Animals
Bacterial Proteins/genetics
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Dependovirus/genetics
Disease Models, Animal
Dystrophin/*genetics
Endonucleases/genetics
Gene Editing/*methods
Genetic Therapy/methods
Genetic Vectors
High-Throughput Nucleotide Sequencing
Male
Mice
Mice, Inbred mdx
Muscle, Skeletal/physiopathology
Muscular Dystrophy, Duchenne/*genetics/*metabolism/*physiopathology/therapy
Mutation
Myocardium
Neuromuscular Diseases/therapy
RNA, Guide
Sequence Deletion

@article {pmid30418590,
year = {2018},
author = {Gordeeva, J and Morozova, N and Sierro, N and Isaev, A and Sinkunas, T and Tsvetkova, K and Matlashov, M and Truncaite, L and Morgan, RD and Ivanov, NV and Siksnys, V and Zeng, L and Severinov, K},
title = {BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gky1125},
pmid = {30418590},
issn = {1362-4962},
abstract = {Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage λ infection, induction of λ prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.},
}

@article {pmid30418108,
year = {2018},
author = {Aung, MS and San, T and San, N and Oo, WM and Ko, PM and Thet, KT and Urushibara, N and Kawaguchiya, M and Sumi, A and Kobayashi, N},
title = {Molecular characterization of Staphylococcus argenteus in Myanmar: identification of novel genotypes/clusters in staphylocoagulase, protein A, alpha-haemolysin and other virulence factors.},
journal = {Journal of medical microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1099/jmm.0.000869},
pmid = {30418108},
issn = {1473-5644},
abstract = {PURPOSE: Staphylococcus argenteus is a novel emerging species of coagulase-positive staphylococcus that is genetically closely related to Staphylococcus aureus. To elucidate the molecular differences in the virulence factors (staphylocoagulase, protein A, alpha-haemolysin, enterotoxin-like toxin and staphylokinase) between these staphylococcal species, S. argenteus that had recently been isolated in Myanmar (five nasal isolates and four clinical isolates) were analysed.

METHODOLOGY: The nucleotide sequences of the virulence factors were determined by PCR and direct sequencing, followed by phylogenetic analysis by mega6 and multiple alignment by clustalw using the published sequence data for S. aureus and S. argenteus.

RESULTS: Six S. argenteus isolates belonged to MLST sequence type (ST) 2250, while others belonged to ST4625, ST2198 and ST2854. The novel staphylocoagulase (coa) genotype XIV and the novel coa-XI subtype (XId) were identified in an ST2198 isolate and all other isolates, respectively. Among the S. argenteus isolates, the protein A and alpha-haemolysin genes showed high sequence identity (96-98 % and >99 %, respectively), while lower identity was observed between S. argenteus and S. aureus (88-91 % and 86 %, respectively), with both species showing phylogenetically distinct clusters. Similar findings were found for the staphylococcal enterotoxin (SE)-like toxin genes selw, selx and sely. In contrast, the staphylokinase genes were almost identical between these two species. All of the coa-XId isolates had a CRISPR/Cas locus at the site of orfX without having SCCmec, whereas an ST2198 isolate lacked this locus.

CONCLUSION: The primary virulence factors (staphylocoagulase, protein A andalpha-haemolysin) as well as the SE-like toxins of S. argenteus were genetically discriminated from those of S. aureus, revealing the presence of the novel coa-type/subtype (coa-IXd, XIV) in S. argenteus.},
}

@article {pmid30413649,
year = {2018},
author = {Li, CJ and Jiang, C and Liu, Y and Bell, T and Ma, W and Ye, Y and Huang, S and Guo, H and Zhang, H and Wang, L and Wang, J and Nomie, K and Zhang, L and Wang, M},
title = {Pleiotropic Action of Novel Bruton's Tyrosine Kinase Inhibitor BGB-3111 in Mantle Cell Lymphoma.},
journal = {Molecular cancer therapeutics},
volume = {},
number = {},
pages = {},
doi = {10.1158/1535-7163.MCT-18-0478},
pmid = {30413649},
issn = {1538-8514},
abstract = {Bruton's tyrosine kinase (BTK) is a key mediator of BCR-dependent cell growth signaling and a clinically effective therapeutic target in mantle cell lymphoma (MCL). The molecular impact of BTK inhibition remains unclear particularly in hematopoietic malignancies. We analyzed the molecular mechanisms of BTK inhibition with the novel inhibitor BGB-3111 (zanubrutinib) in MCL models. The efficacy of BGB-3111 was investigated using growth proliferation/cell viability and apoptosis assays in MCL cell lines and patient-derived xenograft (PDX) MCL cells. The activity and mechanisms of BGB-3111 were further confirmed using a cell line xenograft model, an MCL PDX mouse model, and a human phosphokinase profiler array and reverse phase protein array. Lastly, the mechanisms related to resistance to BTK inhibition were analyzed by creating cell lines with low levels of BTK using CRISPR/Cas 9 genome editing. We found that inhibition of BTK leads to suppression of tumor growth, which was mediated via potent suppression of Akt/mTOR, apoptosis, and metabolic stress. Moreover, targeted disruption of the BTK gene in MCL cells resulted in resistance to BTK inhibition and the emergence of novel survival mechanisms. Our studies suggest a general efficacy of BTK inhibition in MCL and potential drug resistance mechanism via alternative signaling pathways.},
}

@article {pmid30259067,
year = {2018},
author = {Xiang, G and Ren, J and Hai, T and Fu, R and Yu, D and Wang, J and Li, W and Wang, H and Zhou, Q},
title = {Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {75},
number = {24},
pages = {4619-4628},
doi = {10.1007/s00018-018-2917-6},
pmid = {30259067},
issn = {1420-9071},
support = {31471215//National Natural Science Foundation of China/ ; 31422038//National Natural Science Foundation of China/ ; 31471395//National Natural Science Foundation of China/ ; 31501188//National Natural Science Foundation of China/ ; XDA16010205//Strategic Priority Research Program of Chinese Academy of Sciences/ ; 2015AA020307//National High-Tech Research and Development Program/ ; 2016ZX08010001//National Special Key Project for Transgenic Breeding/ ; },
mesh = {Alleles ; Animal Husbandry/methods ; Animals ; Breeding/methods ; *CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Genotype ; Insulin-Like Growth Factor II/*genetics ; *Introns ; Male ; Meat/analysis ; Mutation ; Phenotype ; *Regulatory Sequences, Nucleic Acid ; Swine/*genetics ; },
abstract = {Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR-Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3-3072 site using CRISPR-Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.},
}

Alleles
Animal Husbandry/methods
Animals
Breeding/methods
*CRISPR-Cas Systems
Female
Gene Editing/*methods
Genotype
Insulin-Like Growth Factor II/*genetics
*Introns
Male
Meat/analysis
Mutation
Phenotype
*Regulatory Sequences, Nucleic Acid
Swine/*genetics

@article {pmid29079543,
year = {2017},
author = {Li, X and Xie, Y and Zhu, Q and Liu, YG},
title = {Targeted Genome Editing in Genes and cis-Regulatory Regions Improves Qualitative and Quantitative Traits in Crops.},
journal = {Molecular plant},
volume = {10},
number = {11},
pages = {1368-1370},
doi = {10.1016/j.molp.2017.10.009},
pmid = {29079543},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/*genetics/growth & development ; *Gene Editing ; Genes, Plant/*genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; },
}

CRISPR-Cas Systems/genetics
Crops, Agricultural/*genetics/growth & development
*Gene Editing
Genes, Plant/*genetics
Regulatory Sequences, Nucleic Acid/*genetics

@article {pmid30355950,
year = {2018},
author = {Xu, J and Barone, S and Zahedi, K and Brooks, M and Soleimani, M},
title = {Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {50},
number = {4},
pages = {1361-1375},
doi = {10.1159/000494596},
pmid = {30355950},
issn = {1421-9778},
mesh = {Animals ; Bicarbonates/metabolism ; CRISPR-Cas Systems/genetics ; Diuresis/drug effects ; Dogs ; Furosemide/pharmacology ; In Situ Hybridization ; Kidney/*metabolism ; Kidney Tubules, Collecting/metabolism ; Madin Darby Canine Kidney Cells ; Mice ; Mice, Knockout ; Oligoribonucleotides, Antisense/metabolism ; Plasmids/genetics/metabolism ; Sodium/urine ; Sodium Chloride/metabolism ; Sodium-Bicarbonate Symporters/deficiency/genetics/*metabolism ; },
abstract = {BACKGROUND/AIMS: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney.

METHODS: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice.

RESULTS: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis.

CONCLUSION: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.},
}

Animals
Bicarbonates/metabolism
CRISPR-Cas Systems/genetics
Diuresis/drug effects
Dogs
Furosemide/pharmacology
In Situ Hybridization
Kidney/*metabolism
Kidney Tubules, Collecting/metabolism
Madin Darby Canine Kidney Cells
Mice
Mice, Knockout
Oligoribonucleotides, Antisense/metabolism
Plasmids/genetics/metabolism
Sodium/urine
Sodium Chloride/metabolism
Sodium-Bicarbonate Symporters/deficiency/genetics/*metabolism

@article {pmid28224990,
year = {2017},
author = {Senturk, S and Shirole, NH and Nowak, DG and Corbo, V and Pal, D and Vaughan, A and Tuveson, DA and Trotman, LC and Kinney, JB and Sordella, R},
title = {Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14370},
doi = {10.1038/ncomms14370},
pmid = {28224990},
issn = {2041-1723},
support = {P30 CA045508/CA/NCI NIH HHS/United States ; R01 CA137050/CA/NCI NIH HHS/United States ; U01 CA168409/CA/NCI NIH HHS/United States ; },
mesh = {A549 Cells ; Animals ; CRISPR-Associated Proteins/chemistry/metabolism ; CRISPR-Cas Systems/*genetics ; Fibroblasts/metabolism ; Gene Editing/*methods ; Humans ; Integrases/metabolism ; Lentivirus/metabolism ; Ligands ; Mice ; Protein Domains ; Protein Stability ; RNA, Guide/metabolism ; Tamoxifen/pharmacology ; Time Factors ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes.},
}

A549 Cells
Animals
CRISPR-Associated Proteins/chemistry/metabolism
CRISPR-Cas Systems/*genetics
Fibroblasts/metabolism
Gene Editing/*methods
Humans
Integrases/metabolism
Lentivirus/metabolism
Ligands
Mice
Protein Domains
Protein Stability
RNA, Guide/metabolism
Tamoxifen/pharmacology
Time Factors

@article {pmid28181494,
year = {2017},
author = {Kim, IS and Heilmann, S and Kansler, ER and Zhang, Y and Zimmer, M and Ratnakumar, K and Bowman, RL and Simon-Vermot, T and Fennell, M and Garippa, R and Lu, L and Lee, W and Hollmann, T and Xavier, JB and White, RM},
title = {Microenvironment-derived factors driving metastatic plasticity in melanoma.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14343},
doi = {10.1038/ncomms14343},
pmid = {28181494},
issn = {2041-1723},
support = {DP2 CA186572/CA/NCI NIH HHS/United States ; K08 AR055368/AR/NIAMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Cell Plasticity/genetics ; Cell Proliferation/genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Melanoma/genetics/*pathology ; Models, Biological ; Neoplasm Metastasis ; Phenotype ; *Tumor Microenvironment/genetics ; Zebrafish ; Zebrafish Proteins/metabolism ; },
abstract = {Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITFLO versus proliferative/MITFHI states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITFHI/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation. We screened for microenvironmental factors leading to phenotype switching, and find that EDN3 induces a state that is both proliferative and differentiated. CRISPR-mediated inactivation of EDN3, or its synthetic enzyme ECE2, from the microenvironment abrogates phenotype switching and increases animal survival. These results demonstrate that after metastatic dissemination, the microenvironment provides signals to promote phenotype switching and provide proof that targeting tumour cell plasticity is a viable therapeutic opportunity.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
*Cell Plasticity/genetics
Cell Proliferation/genetics
Gene Expression Profiling
Gene Expression Regulation, Neoplastic
Humans
Melanoma/genetics/*pathology
Models, Biological
Neoplasm Metastasis
Phenotype
*Tumor Microenvironment/genetics
Zebrafish
Zebrafish Proteins/metabolism

@article {pmid29907613,
year = {2018},
author = {Pflueger, C and Tan, D and Swain, T and Nguyen, T and Pflueger, J and Nefzger, C and Polo, JM and Ford, E and Lister, R},
title = {A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs.},
journal = {Genome research},
volume = {28},
number = {8},
pages = {1193-1206},
doi = {10.1101/gr.233049.117},
pmid = {29907613},
issn = {1549-5469},
support = {R01 DA036906/DA/NIDA NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Binding Sites ; CRISPR-Cas Systems/*genetics ; Catalytic Domain/genetics ; Chromatin/genetics ; DNA (Cytosine-5-)-Methyltransferases/*genetics ; DNA Methylation/*genetics ; *Epigenesis, Genetic ; Gene Editing ; Promoter Regions, Genetic ; Protein Binding ; Recombinant Fusion Proteins/*genetics ; },
abstract = {Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.},
}

Binding Sites
CRISPR-Cas Systems/*genetics
Catalytic Domain/genetics
Chromatin/genetics
DNA (Cytosine-5-)-Methyltransferases/*genetics
DNA Methylation/*genetics
*Epigenesis, Genetic
Gene Editing
Promoter Regions, Genetic
Protein Binding
Recombinant Fusion Proteins/*genetics

@article {pmid29720392,
year = {2018},
author = {Yamamoto, M and Nishio, T and Nasrallah, JB},
title = {Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {7},
pages = {2231-2239},
doi = {10.1534/g3.118.200231},
pmid = {29720392},
issn = {2160-1836},
mesh = {Adaptor Protein Complex 2/*metabolism ; Amino Acid Motifs ; Amino Acid Sequence ; Arabidopsis/*genetics/*metabolism ; CRISPR-Cas Systems ; Clathrin/*metabolism ; *Endocytosis ; Gene Expression ; Gene Targeting ; Mutation ; Plant Proteins/chemistry/genetics/metabolism ; Plants, Genetically Modified ; Pollination ; Protein Binding ; Protein Interaction Domains and Motifs ; *Self-Incompatibility in Flowering Plants ; *Signal Transduction ; },
abstract = {Internalization of plasma membrane (PM)-localized ligand-activated receptor kinases and their trafficking to sorting endosomes have traditionally been viewed as functioning primarily in the down-regulation of receptor signaling, but are now considered to be also essential for signaling by some receptors. A major mechanism for internalization of PM proteins is clathrin-mediated endocytosis (CME). CME is mediated by the Adaptor Protein Complex 2 (AP2), which is involved in interaction of the AP2 μ-adaptin subunit with a tyrosine-based Yxxϕ motif located in the cytoplasmic domain of the cargo protein. In this study, we investigated the role of AP2-mediated CME for signaling by the S-locus receptor kinase (SRK), a protein localized in the PM of stigma epidermal cells, which, together with its pollen coat-localized S-locus cysteine-rich (SCR) ligand, functions in the self-incompatibility (SI) response of the Brassicaceae. Using Arabidopsis thaliana plants that were made self-incompatible by transformation with an A. lyrata-derived SRK/SCR gene pair, we tested the effect on SI of site-directed mutations in each of the two Yxxϕ motifs in SRK and of a CRISPR/Cas9-induced null mutation in the AP2 μ-adaptin gene AP2M Both in vitro SRK kinase activity and the in planta SI response were abolished by substitution of tyrosine in one of the two Yxxϕ motifs, but were unaffected by elimination of either the second Yxxϕ motif or AP2M function. Thus, AP2-mediated CME is considered to be unnecessary for SRK signaling in the SI response.},
}

Adaptor Protein Complex 2/*metabolism
Amino Acid Motifs
Amino Acid Sequence
Arabidopsis/*genetics/*metabolism
CRISPR-Cas Systems
Clathrin/*metabolism
*Endocytosis
Gene Expression
Gene Targeting
Mutation
Plant Proteins/chemistry/genetics/metabolism
Plants, Genetically Modified
Pollination
Protein Binding
Protein Interaction Domains and Motifs
*Self-Incompatibility in Flowering Plants
*Signal Transduction

@article {pmid30065322,
year = {2018},
author = {Callaway, E},
title = {CRISPR plants now subject to tough GM laws in European Union.},
journal = {Nature},
volume = {560},
number = {7716},
pages = {16},
doi = {10.1038/d41586-018-05814-6},
pmid = {30065322},
issn = {1476-4687},
mesh = {Agriculture/economics/legislation & jurisprudence/methods ; CRISPR-Cas Systems/*genetics ; *European Union ; *Food, Genetically Modified/economics/standards ; Gene Editing/*legislation & jurisprudence/standards/trends ; Humans ; Mutagenesis ; Plant Breeding/economics/legislation & jurisprudence/methods ; Research/*legislation & jurisprudence/trends ; },
}

Agriculture/economics/legislation & jurisprudence/methods
CRISPR-Cas Systems/*genetics
*European Union
*Food, Genetically Modified/economics/standards
Gene Editing/*legislation & jurisprudence/standards/trends
Humans
Mutagenesis
Plant Breeding/economics/legislation & jurisprudence/methods
Research/*legislation & jurisprudence/trends

@article {pmid29882718,
year = {2018},
author = {Lim, D},
title = {Disruption and development: the evolving CRISPR patent and technology landscape.},
journal = {Pharmaceutical patent analyst},
volume = {7},
number = {4},
pages = {141-145},
doi = {10.4155/ppa-2018-0010},
pmid = {29882718},
issn = {2046-8962},
mesh = {Academies and Institutes ; *CRISPR-Cas Systems ; California ; *Patents as Topic ; Technology ; Universities ; },
}

Academies and Institutes
*CRISPR-Cas Systems
California
*Patents as Topic
Technology
Universities

@article {pmid29718583,
year = {2018},
author = {Hiruta, C and Kakui, K and Tollefsen, KE and Iguchi, T},
title = {Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {23},
number = {6},
pages = {494-502},
doi = {10.1111/gtc.12589},
pmid = {29718583},
issn = {1365-2443},
mesh = {Animals ; Arthropod Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Daphnia/embryology/*genetics/growth & development/physiology ; Deoxyribonuclease I/metabolism ; Embryo, Nonmammalian/cytology/metabolism ; Gene Targeting/*methods ; Genomics ; Mutagenesis ; Phenotype ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D. pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D. pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.},
}

Animals
Arthropod Proteins/genetics/*metabolism
*CRISPR-Cas Systems
Daphnia/embryology/*genetics/growth & development/physiology
Deoxyribonuclease I/metabolism
Embryo, Nonmammalian/cytology/metabolism
Gene Targeting/*methods
Genomics
Mutagenesis
Phenotype
Ribonucleoproteins/genetics/*metabolism

@article {pmid29444903,
year = {2018},
author = {Wei, Y and Yang, L and Zhang, X and Sui, D and Wang, C and Wang, K and Shan, M and Guo, D and Wang, H},
title = {Generation and Characterization of a CYP2C11-Null Rat Model by Using the CRISPR/Cas9 Method.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {46},
number = {5},
pages = {525-531},
doi = {10.1124/dmd.117.078444},
pmid = {29444903},
issn = {1521-009X},
mesh = {Animals ; Aryl Hydrocarbon Hydroxylases/*genetics ; CRISPR-Cas Systems/drug effects/*genetics ; Cytochrome P450 Family 2/*genetics ; Female ; Inactivation, Metabolic/physiology ; Male ; Microsomes, Liver/drug effects ; Models, Animal ; Rats ; Rats, Sprague-Dawley ; Steroid 16-alpha-Hydroxylase/*genetics ; Tolbutamide/pharmacology ; },
abstract = {CYP2C11 is involved in the metabolism of many drugs in rats. To assess the roles of CYP2C11 in physiology and drug metabolism, a CYP2C11-null rat model was generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9method. A 2-base pair insertion was added to exon 6 of CYP2C11 in Sprague-Dawley rats. CYP2C11 was not detected by western blotting in liver microsomes of CYP2C11-null rats. No off-target effects were found at 11 predicted sites of the knockout model. The CYP2C11-null rats were viable and had no obvious abnormalities, with the exception of reduced fertility. Puberty in CYP2C11-null rats appeared to be delayed by ∼20 days, and the average litter size fell by 43%. Tolbutamide was used as a probe in this drug metabolism study. In the liver microsomes of CYP2C11-null rats, the Vmax and intrinsicclearance values decreased by 22% and 47%, respectively, compared with those of wild-type rats. The Km values increased by 47% compared with that of wild types. However, our pharmacokinetics study showed no major differences in any parameters between the two strains, in both males and females. In conclusion, a CYP2C11-null rat model was successfully generated and is a valuable tool to study the in vivo function of CYP2C11.},
}

Animals
Aryl Hydrocarbon Hydroxylases/*genetics
CRISPR-Cas Systems/drug effects/*genetics
Cytochrome P450 Family 2/*genetics
Female
Inactivation, Metabolic/physiology
Male
Microsomes, Liver/drug effects
Models, Animal
Rats
Rats, Sprague-Dawley
Steroid 16-alpha-Hydroxylase/*genetics
Tolbutamide/pharmacology

@article {pmid28418037,
year = {2017},
author = {Yang, Y and Xiong, S and Cai, B and Luo, H and Dong, E and Li, Q and Ji, G and Zhao, C and Wen, Y and Wei, Y and Yang, H},
title = {Mitochondrial C11orf83 is a potent Antiviral Protein Independent of interferon production.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44303},
doi = {10.1038/srep44303},
pmid = {28418037},
issn = {2045-2322},
mesh = {2',5'-Oligoadenylate Synthetase/genetics/*immunology ; Animals ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*immunology ; Cercopithecus aethiops ; Endoribonucleases/genetics/*immunology ; Gene Deletion ; Gene Expression Regulation ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Hep G2 Cells ; *Host-Pathogen Interactions ; Human Umbilical Vein Endothelial Cells ; Humans ; Immunity, Innate ; Interferon-gamma/genetics/immunology ; Luciferases/genetics/metabolism ; Mitochondria/genetics/*immunology ; Signal Transduction ; Vero Cells ; Vesicular stomatitis Indiana virus/genetics/*immunology ; Virus Replication ; },
abstract = {Mitochondria have a central position in innate immune response via the adaptor protein MAVS in mitochondrial outer membrane to limit viral replication by inducing interferon production. Here, we reported that C11orf83, a component of complex III of electronic transfer chain in mitochondrial inner membrane, was a potent antiviral protein independent of interferon production. C11orf83 expression significantly increased in response to viral infection, and endows cells with stronger capability of inhibiting viral replication. Deletion of C11orf83 permits viral replication easier and cells were more vulnerable to viral killing. These effects mainly were mediated by triggering OAS3-RNase L system. C11orf83 overexpression induced higher transcription of OAS3, and knockdown either OAS3 or RNase L impaired the antiviral capability of C11orf83. Interestingly, the signaling from C11orf83 to OAS3-RNase L was independent of interferon production. Thus, our findings suggested a new antiviral mechanism by bridging cell metabolic machinery component with antiviral effectors.},
}

2',5'-Oligoadenylate Synthetase/genetics/*immunology
Animals
CRISPR-Cas Systems
Carrier Proteins/genetics/*immunology
Cercopithecus aethiops
Endoribonucleases/genetics/*immunology
Gene Deletion
Gene Expression Regulation
Genes, Reporter
Green Fluorescent Proteins/genetics/metabolism
HEK293 Cells
Hep G2 Cells
*Host-Pathogen Interactions
Human Umbilical Vein Endothelial Cells
Humans
Immunity, Innate
Interferon-gamma/genetics/immunology
Luciferases/genetics/metabolism
Mitochondria/genetics/*immunology
Signal Transduction
Vero Cells
Vesicular stomatitis Indiana virus/genetics/*immunology
Virus Replication

@article {pmid28287132,
year = {2017},
author = {Wrona, D and Siler, U and Reichenbach, J},
title = {CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44187},
doi = {10.1038/srep44187},
pmid = {28287132},
issn = {2045-2322},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Genetic Therapy/*methods ; *Genetic Vectors ; *Granulomatous Disease, Chronic/genetics/metabolism/pathology/therapy ; HL-60 Cells ; Humans ; NADPH Oxidases/genetics/metabolism ; Sequence Deletion ; },
abstract = {Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.},
}

Base Sequence
*CRISPR-Cas Systems
Genetic Therapy/*methods
*Genetic Vectors
*Granulomatous Disease, Chronic/genetics/metabolism/pathology/therapy
HL-60 Cells
Humans
NADPH Oxidases/genetics/metabolism
Sequence Deletion

@article {pmid28281657,
year = {2017},
author = {Pazhakh, V and Clark, S and Keightley, MC and Lieschke, GJ},
title = {A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44455},
doi = {10.1038/srep44455},
pmid = {28281657},
issn = {2045-2322},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo, Nonmammalian ; Exons ; Gene Editing/*methods ; Gene Expression ; Granulocyte Colony-Stimulating Factor/*genetics/immunology ; Haploinsufficiency ; Heterozygote ; Humans ; Kidney/immunology/*pathology ; Leukocyte Count ; Morpholinos/genetics/metabolism ; Neutropenia/*congenital/genetics/immunology/pathology ; Neutrophils/immunology/*pathology ; Phenotype ; Receptors, Colony-Stimulating Factor/antagonists & inhibitors/deficiency/*genetics/immunology ; Zebrafish ; },
abstract = {Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration. We created an allelic series of zebrafish csf3r mutants by CRISPR/Cas9 mutagenesis targeting csf3r exon 2. Biallelic csf3r mutant embryos are viable and have normal early survival, despite a substantial reduction of their neutrophil population size, and normal macrophage abundance. Heterozygotes have a haploinsufficiency phenotype with an intermediate reduction in neutrophil numbers. csf3r mutants are viable as adults, with a 50% reduction in tissue neutrophil density and a substantial reduction in the number of myeloid cells in the kidney marrow. These csf3r mutants are a new animal model of human CSF3R-dependent congenital neutropenia. Furthermore, they will be valuable for studying the impact of neutrophil loss in the context of other zebrafish disease models by providing a genetically stable, persistent, reproducible neutrophil deficiency state throughout life.},
}

Animals
Base Sequence
CRISPR-Cas Systems
Disease Models, Animal
Embryo, Nonmammalian
Exons
Gene Editing/*methods
Gene Expression
Granulocyte Colony-Stimulating Factor/*genetics/immunology
Haploinsufficiency
Heterozygote
Humans
Kidney/immunology/*pathology
Leukocyte Count
Morpholinos/genetics/metabolism
Neutropenia/*congenital/genetics/immunology/pathology
Neutrophils/immunology/*pathology
Phenotype
Receptors, Colony-Stimulating Factor/antagonists & inhibitors/deficiency/*genetics/immunology
Zebrafish

@article {pmid30397647,
year = {2018},
author = {Chatterjee, P and Jakimo, N and Jacobson, JM},
title = {Minimal PAM specificity of a highly similar SpCas9 ortholog.},
journal = {Science advances},
volume = {4},
number = {10},
pages = {eaau0766},
doi = {10.1126/sciadv.aau0766},
pmid = {30397647},
issn = {2375-2548},
abstract = {RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5'-NNG-3' PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.},
}

@article {pmid30397343,
year = {2018},
author = {Özcan, A and Pausch, P and Linden, A and Wulf, A and Schühle, K and Heider, J and Urlaub, H and Heimerl, T and Bange, G and Randau, L},
title = {Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41564-018-0274-8},
pmid = {30397343},
issn = {2058-5276},
abstract = {Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein-protein and protein-RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR-Cas systems and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided effector complexes.},
}

@article {pmid29950430,
year = {2018},
author = {Chen, X and Liao, S and Huang, X and Xu, T and Feng, X and Guang, S},
title = {Targeted Chromosomal Rearrangements via Combinatorial Use of CRISPR/Cas9 and Cre/LoxP Technologies in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {8},
pages = {2697-2707},
doi = {10.1534/g3.118.200473},
pmid = {29950430},
issn = {2160-1836},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; *Chromosome Inversion ; Gene Editing/*methods ; Gene Targeting/*methods ; Integrases/genetics/metabolism ; },
abstract = {Rearranged chromosomes have been applied to construct genetic balancers to manipulate essential genes in C. elegans Although much effort has been put into constructing balancer chromosomes, approximately 6% (map units) of the C. elegans genome has not been covered, and this area lies mostly in pairing centers (PCs). Here, we developed a method for conditional chromosomal engineering through combinatorial use of the CRISPR/Cas9 and Cre/LoxP technologies. Functional DNA fragments containing LoxP sequences were inserted into designated genomic loci using a modified counterselection (cs)-CRISPR method. Then, heat-shock-induced Cre recombinase induced an inversion of the chromosomal region between the two LoxP sites. The chromosomal inversions were subsequently detected by the appearance of pharyngeal GFP. Through this method, we have successfully generated several chromosomal inversion lines, providing valuable resources for studying essential genes in pairing centers.},
}

Animals
*CRISPR-Cas Systems
Caenorhabditis elegans/genetics
*Chromosome Inversion
Gene Editing/*methods
Gene Targeting/*methods
Integrases/genetics/metabolism

@article {pmid29934375,
year = {2018},
author = {Ewen-Campen, B and Perrimon, N},
title = {ovoD Co-selection: A Method for Enriching CRISPR/Cas9-Edited Alleles in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {8},
pages = {2749-2756},
doi = {10.1534/g3.118.200498},
pmid = {29934375},
issn = {2160-1836},
support = {R01 GM084947/GM/NIGMS NIH HHS/United States ; F32 GM113395/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Drosophila/*genetics ; Female ; Gene Editing/methods ; Gene Targeting/*methods ; Oogonia/metabolism ; Phenotype ; *Selection, Genetic ; },
abstract = {Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1 In this system ("ovoD co-selection"), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovoD co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.},
}

Alleles
Animals
*CRISPR-Cas Systems
Drosophila/*genetics
Female
Gene Editing/methods
Gene Targeting/*methods
Oogonia/metabolism
Phenotype
*Selection, Genetic

@article {pmid29884615,
year = {2018},
author = {Pauwels, L and De Clercq, R and Goossens, J and Iñigo, S and Williams, C and Ron, M and Britt, A and Goossens, A},
title = {A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {8},
pages = {2603-2615},
doi = {10.1534/g3.118.200046},
pmid = {29884615},
issn = {2160-1836},
mesh = {Arabidopsis/*genetics ; Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA-Directed DNA Polymerase/genetics/metabolism ; Gene Editing/*methods ; Genomics/*methods ; *Mutagenesis ; RNA, Guide/*genetics/metabolism ; },
abstract = {Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase θ (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.},
}

Arabidopsis/*genetics
Arabidopsis Proteins/genetics/metabolism
CRISPR-Cas Systems
DNA End-Joining Repair
DNA-Directed DNA Polymerase/genetics/metabolism
Gene Editing/*methods
Genomics/*methods
*Mutagenesis
RNA, Guide/*genetics/metabolism

@article {pmid29703785,
year = {2018},
author = {Zhang, XR and He, JB and Wang, YZ and Du, LL},
title = {A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {6},
pages = {2067-2077},
doi = {10.1534/g3.118.200164},
pmid = {29703785},
issn = {2160-1836},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Cloning, Molecular ; DNA Repair/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques ; Point Mutation/genetics ; Schizosaccharomyces/*genetics ; Sequence Deletion ; Temperature ; },
abstract = {The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.},
}

Base Sequence
CRISPR-Cas Systems/*genetics
Cloning, Molecular
DNA Repair/genetics
Gene Editing/*methods
Gene Knock-In Techniques
Point Mutation/genetics
Schizosaccharomyces/*genetics
Sequence Deletion
Temperature

@article {pmid29542087,
year = {2018},
author = {Gandhi, S and Razy-Krajka, F and Christiaen, L and Stolfi, A},
title = {CRISPR Knockouts in Ciona Embryos.},
journal = {Advances in experimental medicine and biology},
volume = {1029},
number = {},
pages = {141-152},
doi = {10.1007/978-981-10-7545-2_13},
pmid = {29542087},
issn = {0065-2598},
support = {K99 HD084814/HD/NICHD NIH HHS/United States ; R00 HD084814/HD/NICHD NIH HHS/United States ; R01 GM096032/GM/NIGMS NIH HHS/United States ; R01 HL108643/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Ciona intestinalis/embryology/*genetics ; Electroporation ; Embryo, Nonmammalian/cytology/metabolism ; Gene Knockout Techniques/*methods ; INDEL Mutation ; Mutagenesis ; Plasmids ; RNA, Guide/administration & dosage/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a revolutionary tool for fast and efficient targeted gene knockouts and genome editing in almost any organism. The laboratory model tunicate Ciona is no exception. Here, we describe our latest protocol for the design, implementation, and evaluation of successful CRISPR/Cas9-mediated gene knockouts in somatic cells of electroporated Ciona embryos. Using commercially available reagents, publicly accessible plasmids, and free web-based software applications, any Ciona researcher can easily knock out any gene of interest in their favorite embryonic cell lineage.},
}

Animals
CRISPR-Cas Systems
Ciona intestinalis/embryology/*genetics
Electroporation
Embryo, Nonmammalian/cytology/metabolism
Gene Knockout Techniques/*methods
INDEL Mutation
Mutagenesis
Plasmids
RNA, Guide/administration & dosage/genetics

@article {pmid28256588,
year = {2017},
author = {Li, C and Unver, T and Zhang, B},
title = {A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.).},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {43902},
doi = {10.1038/srep43902},
pmid = {28256588},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; DNA, Plant/genetics/metabolism ; Gene Targeting/*methods ; Gossypium/*genetics ; Recombination, Genetic ; },
abstract = {The complex allotetraploid genome is one of major challenges in cotton for repressing gene expression. Developing site-specific DNA mutation is the long-term dream for cotton breeding scientists. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as a robust biotechnology for targeted-DNA mutation. In this study, two sgRNAs, GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, were designed in the identical genomic regions of GhMYB25-like A and GhMYB25-like D, which were encoded by cotton A subgenome and the D subgenome, respectively, was assembled to direct Cas9-mediated allotetraploid cotton genome editing. High proportion (14.2-21.4%) CRISPR/Cas9-induced specific truncation events, either from GhMYB25-like A DNA site or from GhMYB25-like D DNA site, were detected in 50% examined transgenic cotton through PCR amplification assay and sequencing analyses. Sequencing results also demonstrated that 100% and 98.8% mutation frequency were occurred on GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2 target site respectively. The off-target effect was evaluated by sequencing two putative off-target sites, which have 3 and 1 mismatched nucleotides with GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, respectively; all the examined samples were not detected any off-target-caused mutation events. Thus, these results demonstrated that CRISPR/Cas9 is qualified for generating DNA level mutations on allotetraploid cotton genome with high-efficiency and high-specificity.},
}

*CRISPR-Cas Systems
DNA, Plant/genetics/metabolism
Gene Targeting/*methods
Gossypium/*genetics
Recombination, Genetic

@article {pmid28240315,
year = {2017},
author = {Sanz, MA and González Almela, E and Carrasco, L},
title = {Translation of Sindbis Subgenomic mRNA is Independent of eIF2, eIF2A and eIF2D.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {43876},
doi = {10.1038/srep43876},
pmid = {28240315},
issn = {2045-2322},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Eukaryotic Cells/cytology/virology ; Eukaryotic Initiation Factor-2/genetics/*metabolism ; Humans ; Phosphorylation ; *Protein Biosynthesis ; RNA Interference ; RNA, Messenger/*genetics ; RNA, Viral/*genetics ; Sindbis Virus/*genetics/physiology ; Viral Proteins/genetics/metabolism ; },
abstract = {Translation of Sindbis virus subgenomic mRNA (sgmRNA) can occur after inactivation of eIF2 by phosphorylation in mammalian cells. Several studies have suggested that eIF2 can be replaced by eIF2A or eIF2D. HAP1 human cell lines knocked-out for eIF2A, eIF2D or both by CRISPR/Cas9 genome engineering were compared with wild-type (WT) cells to test the potential role of eIF2A and eIF2D in translation. Sindbis virus infection was comparable between the four cell lines. Moreover, synthesis of viral proteins during late stage infection was similar in all four cell lines despite the fact that eIF2α became phosphorylated. These findings demonstrate that eIF2A and eIF2D are not required for the translation of sgmRNA when eIF2α is phosphorylated. Moreover, silencing of eIF2A or eIF2D by transfection of the corresponding siRNAs in HAP1 WT, HAP1-eIF2A- and HAP1-eIF2D- cells had little effect on the synthesis of viral proteins late in infection. Modification of AUGi to other codons in sgmRNA failed to abrogate translation. Sindbis virus replicons containing these sgmRNA variants could still direct the synthesis of viral proteins. No significant differences were found between the cell lines assayed, suggesting that neither eIF2A nor eIF2D are involved in the translation of this sgmRNA bearing non-AUG codons.},
}

Base Sequence
CRISPR-Cas Systems
Cell Line
Eukaryotic Cells/cytology/virology
Eukaryotic Initiation Factor-2/genetics/*metabolism
Humans
Phosphorylation
*Protein Biosynthesis
RNA Interference
RNA, Messenger/*genetics
RNA, Viral/*genetics
Sindbis Virus/*genetics/physiology
Viral Proteins/genetics/metabolism

@article {pmid30394685,
year = {2018},
author = {Li, P and Fu, X and Zhang, L and Li, S},
title = {CRISPR/Cas-based screening of a gene activation library in Saccharomyces cerevisiae identifies a crucial role of OLE1 in thermotolerance.},
journal = {Microbial biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1751-7915.13333},
pmid = {30394685},
issn = {1751-7915},
support = {21506113//National Natural Science Foundation of China/ ; 31470229//National Natural Science Foundation of China/ ; 31600067//National Natural Science Foundation of China/ ; 2016YFE0108500//National Key R&D Program of China/ ; 2017T100064//China Postdoctoral Science Foundation/ ; 2015M581074//China Postdoctoral Science Foundation/ ; },
abstract = {CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) screening has been proved to be an efficient method to study functional genomics from yeast to human. In this study, we report the development of a focused CRISPR/Cas-based gene activation library in Saccharomyces cerevisiae and its application in gene identification based on functional screening towards improved thermotolerance. The gene activation library was subjected to screening at 42°C, and the same library cultured at 30°C was set as a control group. After five successive subcultures, five clones were randomly picked from the libraries cultured at 30 and 42°C, respectively. The five clones selected at 30°C contain the specificity sequences of five different single guide RNAs, whereas all the five clones selected at 42°C contain the specificity sequence of one sgRNA that targets the promoter region of OLE1. A crucial role of OLE1 in thermotolerance was identified: the overexpression of OLE1 increased fatty acid unsaturation, and thereby helped counter lipid peroxidation caused by heat stress, rendering the yeast thermotolerant. This study described the application of CRISPR/Cas-based gene activation screening with an example of thermotolerant yeast screening, demonstrating that this method can be used to identify functional genes in yeast.},
}

@article {pmid30394652,
year = {2018},
author = {Borton, MA and Daly, RA and O'Banion, B and Hoyt, DW and Marcus, DN and Welch, S and Hastings, SS and Meulia, T and Wolfe, RA and Booker, AE and Sharma, S and Cole, DR and Wunch, K and Moore, JD and Darrah, TH and Wilkins, MJ and Wrighton, KC},
title = {Comparative genomics and physiology of the genus Methanohalophilus, a prevalent methanogen in hydraulically fractured shale.},
journal = {Environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1462-2920.14467},
pmid = {30394652},
issn = {1462-2920},
abstract = {About 60% of natural gas production in the United States comes from hydraulic fracturing of unconventional reservoirs, such as shales or organic-rich micrites. This process inoculates and enriches for halotolerant microorganisms in these reservoirs over time, resulting in a saline ecosystem that includes methane producing archaea. Here, we survey the biogeography of methanogens across unconventional reservoirs, and report that members of genus Methanohalophilus are recovered from every hydraulically fractured unconventional reservoir sampled by metagenomics. We provide the first genomic sequencing of three isolate genomes, as well as two metagenome assembled genomes. Utilizing six other previously sequenced genomes, we perform comparative analysis of the 11 genomes representing this genus. This genomic investigation revealed distinctions between surface and subsurface derived genomes that are consistent with constraints encountered in each environment. Genotypic differences were also uncovered between isolate genomes recovered from the same well, suggesting niche partitioning among closely related strains. These genomic substrate utilization predictions were then confirmed by physiological investigation. Fine-scale microdiversity was observed in CRISPR-Cas systems of Methanohalophilus, with genomes from geographically distinct unconventional reservoirs sharing spacers targeting the same viral population. These findings have implications for augmentation strategies resulting in enhanced biogenic methane production in hydraulically fractured unconventional reservoirs. This article is protected by copyright. All rights reserved.},
}

@article {pmid30394044,
year = {2018},
author = {Ali, S and Park, SK and Kim, WC},
title = {The pragmatic introduction and expression of microbial transgenes in plants.},
journal = {Journal of microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.4014/jmb.1808.08029},
pmid = {30394044},
issn = {1738-8872},
abstract = {Several genetic strategies have been proposed for the successful transformation and expression of microbial transgenes in model and crop plants. Here, we bring into focus the prominent applications of microbial transgenes in plants for the development of disease resistance; mitigation of stress conditions; augmentation of food quality; and use of plants as "bioreactors" for the production of recombinant proteins, industrially important enzymes, vaccines, antimicrobial compounds, and other valuable secondary metabolites. We discuss the applicable and cost-effective approaches of transgenesis in different plants, as well as the limitations thereof. We subsequently present the contemporary developments in targeted genome editing systems that have facilitated the process of genetic modification and manifested stable and consumer-friendly genetically modified plants and their products. Finally, this article presents the different approaches and demonstrates the introduction and expression of microbial transgenes for the improvement of plant resistance to pathogens and abiotic stress conditions and the production of valuable compounds, together with the promising research progress in targeted genome editing technology. We include a special discussion on the highly efficient CRISPR-Cas system helpful in microbial transgene editing in plants.},
}

@article {pmid30393777,
year = {2018},
author = {Zhang, F and Zhao, S and Ren, C and Zhu, Y and Zhou, H and Lai, Y and Zhou, F and Jia, Y and Zheng, K and Huang, Z},
title = {CRISPRminer is a knowledge base for exploring CRISPR-Cas systems in microbe and phage interactions.},
journal = {Communications biology},
volume = {1},
number = {},
pages = {180},
doi = {10.1038/s42003-018-0184-6},
pmid = {30393777},
issn = {2399-3642},
abstract = {CRISPR-Cas systems not only play key roles in prokaryotic acquired immunity, but can also be adapted as powerful genome editing tools. Understanding the native role of CRISPR-Cas systems in providing adaptive immunity can lead to new CRISPR-based technologies. Here, we develop CRISPRminer, a knowledge base and web server to comprehensively collect and investigate the knowledge of CRISPR-Cas systems and generate instructive annotations, including CRISPR arrays and Cas protein annotation, CRISPR-Cas system classification, self-targeting events detection, microbe-phage interaction inference, and anti-CRISPR annotation. CRISPRminer is user-friendly and freely available at http://www.microbiome-bigdata.com/CRISPRminer.},
}

@article {pmid30393194,
year = {2018},
author = {Hussain, W and Mahmood, T and Hussain, J and Ali, N and Shah, T and Qayyum, S and Khan, I},
title = {CRISPR/Cas system: A game changing genome editing technology, to treat human genetic diseases.},
journal = {Gene},
volume = {685},
number = {},
pages = {70-75},
doi = {10.1016/j.gene.2018.10.072},
pmid = {30393194},
issn = {1879-0038},
abstract = {Genes, are the functional units of heredity that used as an instructors to make proteins either to become the functional or structural part of the cell. Hence, the proteins get more attention because most of the life functions depends on it. Any mutation or alteration in genome sequences results in complete loss of function or formation of abnormal protein which leads to hereditary disorder. Gene therapy on the other hand, used as a remedy, a process that make correction in the gene which is responsible for genomic disorders. The treatment of disease state depends on the understanding of their genetic basis. While, numerous molecular genome editing tools have been developed and are being utilized to translate the abstract of gene therapy into reality, but the problem is still a mystery. The genome editing molecular scissors can be applied to dictate the selected genetic products that can have the therapeutic power. Thus, editing the specific sequences depends on the type of strategies being used by a molecule such is HDR or NHEJ. CRISPR/Cas9 editing technology can use in disease model to study the genitival disorders. One side the CRISPR technology seemed to be extremely accurate but on the other side it has some harmful effects i.e. Cas9 proteins sometimes cuts the similar sequences other than the specific targeted and Off-targeting Sequences etc. Urgent attention and improvement are needed for various implication of CRISPR/Cas9 technology, including the delivery, precision and control over the mention system. This review presents the current scenario of genome editing in vivo and its implications for the future of human genetic disease treatment as well as genome throughput potency.},
}

@article {pmid30391232,
year = {2018},
author = {Chen, H and Li, Y and Du, C and Li, Y and Zhao, J and Zheng, X and Mao, Q and Xia, H},
title = {Aptazyme-mediated direct modulation of post-transcriptional sgRNA level for conditional genome editing and gene expression.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2018.10.011},
pmid = {30391232},
issn = {1873-4863},
abstract = {RNA-guided endonuclease Cas9 derived from microbial CRISPR-Cas adaptive immune systems is a powerful tool for genome editing, which has been widely used in eukaryotic systems, prokaryotic systems, and plants. However, the off-target effects caused by Cas9/sgRNA remain a major concern. Currently, the efforts to reduce the off-target effects mainly focus on improving the targeting specificity of sgRNA/Cas9, regulating the activity of the Cas9 protein or the sgRNA, and controlling the time window of their expression. In this study, a novel system was established to regulate the post-transcriptional sgRNA level by small molecule-controlled aptazyme. This system was shown to reduce the off-target effects caused by Cas9/sgRNA, while enabling precise temporal control over gene editing and regulatory activity. This new system could provide a potentially safer and more powerful tool for genome editing and therapeutic application.},
}

@article {pmid30389787,
year = {2018},
author = {Lynch, JM and Li, B and Katoli, P and Xiang, C and Leehy, B and Rangaswamy, N and Saenz-Vash, V and Wang, YK and Lei, H and Nicholson, TB and Meredith, E and Rice, D and Prasanna, G and Chen, A},
title = {Binding of a glaucoma-associated myocilin variant to the αB-crystallin chaperone impedes protein clearance in trabecular meshwork cells.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA118.004325},
pmid = {30389787},
issn = {1083-351X},
abstract = {Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of wild-type (wt) MYOC has remained elusive and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum (ER), and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution which is the equivalent of the pathologic human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaparone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.},
}

@article {pmid30388811,
year = {2018},
author = {Dion, MB and Labrie, SJ and Shah, SA and Moineau, S},
title = {CRISPRStudio: A User-Friendly Software for Rapid CRISPR Array Visualization.},
journal = {Viruses},
volume = {10},
number = {11},
pages = {},
doi = {10.3390/v10110602},
pmid = {30388811},
issn = {1999-4915},
support = {143924//Canadian Institutes of Health Research/Canada ; 259257//Fonds de Recherche du Québec - Nature et Technologies/ ; 950-227645//Canada Research Chairs/ ; RGPIN/05132-2014//Natural Sciences and Engineering Research Council of Canada/ ; },
abstract = {The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly.},
}

@article {pmid30285624,
year = {2018},
author = {Howells, RM and Craze, M and Bowden, S and Wallington, EJ},
title = {Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9.},
journal = {BMC plant biology},
volume = {18},
number = {1},
pages = {215},
doi = {10.1186/s12870-018-1433-z},
pmid = {30285624},
issn = {1471-2229},
support = {BB/J019356/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Agrobacterium/genetics ; *CRISPR-Cas Systems ; DNA, Bacterial ; Gene Editing/*methods ; Genome, Plant ; Hordeum/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Transformation, Genetic ; Triticum/*genetics ; },
abstract = {BACKGROUND: The use of CRISPR/Cas9 systems could prove to be a valuable tool in crop research, providing the ability to fully knockout gene function in complex genomes or to precisely adjust gene function by knockout of individual alleles.

RESULTS: We compare gene editing in hexaploid wheat (Triticum aestivum) with diploid barley (Hordeum vulgare), using a combination of single genome and tri-genome targeting. High efficiency gene editing, 11-17% for single genome targeted guides and 5% for tri-genome targeted guides, was achieved in wheat using stable Agrobacterium-mediated transformation. Gene editing in wheat was shown to be predominantly heterozygous, edits were inherited in a Mendelian fashion over multiple generations and no off-target effects were observed. Comparison of editing between the two species demonstrated that more stable, heritable edits were produced in wheat, whilst barley exhibited continued and somatic editing.

CONCLUSION: Our work shows the potential to obtain stable edited transgene-free wheat lines in 36 weeks through only two generations and that targeted mutagenesis of individual homeologues within the wheat genome is achievable with a modest amount of effort, and without off-target mutations or the need for lengthy crossing strategies.},
}

Agrobacterium/genetics
*CRISPR-Cas Systems
DNA, Bacterial
Gene Editing/*methods
Genome, Plant
Hordeum/genetics
Plant Breeding/methods
Plants, Genetically Modified/genetics
Transformation, Genetic
Triticum/*genetics

@article {pmid30006230,
year = {2018},
author = {Carroll, D},
title = {p53 Throws CRISPR a Curve.},
journal = {Trends in pharmacological sciences},
volume = {39},
number = {9},
pages = {783-784},
doi = {10.1016/j.tips.2018.06.005},
pmid = {30006230},
issn = {1873-3735},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Tumor Suppressor Protein p53/genetics ; },
abstract = {The efficacy of the powerful CRISPR-Cas9 genome-editing platform depends on DNA repair activities in the cells being targeted. Two new papers show that the low efficiency of targeting in some primary human cell lines is the result of p53-dependent cell arrest in response to the Cas9-induced break. This limitation must be overcome for some anticipated therapies.},
}

*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Tumor Suppressor Protein p53/genetics

@article {pmid29991671,
year = {2018},
author = {Cho, LH and Yoon, J and Wai, AH and An, G},
title = {Histone Deacetylase 701 (HDT701) Induces Flowering in Rice by Modulating Expression of OsIDS1.},
journal = {Molecules and cells},
volume = {41},
number = {7},
pages = {665-675},
doi = {10.14348/molcells.2018.0148},
pmid = {29991671},
issn = {0219-1032},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Chromatin/metabolism ; Circadian Rhythm/genetics ; Flowers/*enzymology/*physiology ; *Gene Expression Regulation, Plant ; Histone Deacetylases/chemistry/genetics/*metabolism ; Models, Biological ; Mutation/genetics ; Oryza/*enzymology/*genetics/physiology ; Photoperiod ; Plant Leaves/genetics ; Plant Proteins/chemistry/genetics/*metabolism ; },
abstract = {Rice is a facultative short-day (SD) plant in which flowering is induced under SD conditions or by other environmental factors and internal genetic programs. Overexpression of Histone Deacetylase 701 (HDT701) accelerates flowering in hybrid rice. In this study, mutants defective in HDT701 flowered late under both SD and long-day conditions. Expression levels of florigens Heading date 3a (Hd3a) and Rice Flowering Locus T1 (RFT1), and their immediate upstream floral activator Early heading date 1 (Ehd1), were significantly decreased in the hdt701 mutants, indicating that HDT701 functions upstream of Ehd1 in controlling flowering time. Transcript levels of OsINDETERMINATE SPIKELET 1 (OsIDS1), an upstream repressor of Ehd1, were significantly increased in the mutants while those of OsGI and Hd1 were reduced. Chromatin-immunoprecipitation assays revealed that HDT701 directly binds to the promoter region of OsIDS1. These results suggest that HDT701 induces flowering by suppressing OsIDS1.},
}

Base Sequence
CRISPR-Cas Systems/genetics
Chromatin/metabolism
Circadian Rhythm/genetics
Flowers/*enzymology/*physiology
*Gene Expression Regulation, Plant
Histone Deacetylases/chemistry/genetics/*metabolism
Models, Biological
Mutation/genetics
Oryza/*enzymology/*genetics/physiology
Photoperiod
Plant Leaves/genetics
Plant Proteins/chemistry/genetics/*metabolism

@article {pmid29964176,
year = {2018},
author = {Fernandez, JP and Vejnar, CE and Giraldez, AJ and Rouet, R and Moreno-Mateos, MA},
title = {Optimized CRISPR-Cpf1 system for genome editing in zebrafish.},
journal = {Methods (San Diego, Calif.)},
volume = {150},
number = {},
pages = {11-18},
doi = {10.1016/j.ymeth.2018.06.014},
pmid = {29964176},
issn = {1095-9130},
abstract = {The impact of the CRISPR-Cas biotechnological systems has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient RNA-guided endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that (i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and (ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37 °C. Here, we describe a step-by-step protocol on how to easily design and generate crRNAs in vitro, purify recombinant Cpf1 proteins, and assemble ribonucleoprotein complexes to carry out efficient mutagenesis in zebrafish in a constitutive and temperature-controlled manner. Finally, we explain how to induce Cpf1-mediated homology-directed repair using single-stranded DNA oligonucleotides. In summary, this protocol includes the steps to efficiently modify the zebrafish genome and other ectothermic organisms using the CRISPR-Cpf1 system.},
}

@article {pmid29950431,
year = {2018},
author = {Xu, Y and Wang, Y and Song, Y and Deng, J and Chen, M and Ouyang, H and Lai, L and Li, Z},
title = {Generation and Phenotype Identification of PAX4 Gene Knockout Rabbit by CRISPR/Cas9 System.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {8},
pages = {2833-2840},
doi = {10.1534/g3.118.300448},
pmid = {29950431},
issn = {2160-1836},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Gene Expression ; Gene Knockout Techniques ; Gene Order ; Gene Targeting ; Genetic Vectors ; Immunohistochemistry ; Mutation ; Paired Box Transcription Factors/*deficiency/*genetics/metabolism ; *Phenotype ; RNA, Guide ; Rabbits ; },
abstract = {Paired-homeodomain transcription factor 4 (PAX4) gene encodes a transcription factor which plays an important role in the generation, differentiation, development, and survival of insulin-producing β-cells during mammalian pancreas development. PAX4 is a key diabetes mellitus (DM) susceptibility gene, which is associated with many different types of DM, including T1DM, T2DM, maturity onset diabetes of the young 9 (MODY9) and ketosis prone diabetes. In this study, a novel PAX4 gene knockout (KO) model was generated through co-injection of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) mRNA/sgRNA into rabbit zygotes. Typical phenotypes of growth retardation, persistent hyperglycemia, decreased number of insulin-producing β cells and increased number of glucagon-producing α cells were observed in the homozygous PAX4 KO rabbits. Furthermore, DM associated phenotypes including diabetic nephropathy, hepatopathy, myopathy and cardiomyopathy were also observed in the homozygous PAX4 KO rabbits but not in the wild type (WT) controls and the heterozygous PAX4 KO rabbits. In summary, this is the first PAX4 gene KO rabbit model generated by CRISPR/Cas9 system. This novel rabbit model may provide a new platform for function study of PAX4 gene in rabbit and gene therapy of human DM in clinical trails.},
}

Animals
Animals, Genetically Modified
*CRISPR-Cas Systems
Gene Expression
Gene Knockout Techniques
Gene Order
Gene Targeting
Genetic Vectors
Immunohistochemistry
Mutation
Paired Box Transcription Factors/*deficiency/*genetics/metabolism
*Phenotype
RNA, Guide
Rabbits

@article {pmid29886029,
year = {2018},
author = {Luo, J and Lu, L and Gu, Y and Huang, R and Gui, L and Li, S and Qi, X and Zheng, W and Chao, T and Zheng, Q and Liang, Y and Zhang, L},
title = {Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion.},
journal = {Journal of biotechnology},
volume = {281},
number = {},
pages = {11-20},
doi = {10.1016/j.jbiotec.2018.06.308},
pmid = {29886029},
issn = {1873-4863},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Cricetulus ; DNA ; Gene Editing/*methods ; Humans ; Luminescent Proteins/genetics ; Mice ; Sequence Deletion ; Streptococcus pyogenes/genetics ; },
abstract = {Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection.},
}

Animals
CHO Cells
*CRISPR-Cas Systems
Cell Line
Cell Line, Tumor
Cricetulus
DNA
Gene Editing/*methods
Humans
Luminescent Proteins/genetics
Mice
Sequence Deletion
Streptococcus pyogenes/genetics

@article {pmid29844549,
year = {2018},
author = {Eisenstein, M},
title = {CRISPR takes on Huntington's disease.},
journal = {Nature},
volume = {557},
number = {7707},
pages = {S42-S43},
doi = {10.1038/d41586-018-05177-y},
pmid = {29844549},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; *Gene Editing ; Genetic Therapy/*methods ; Humans ; Huntingtin Protein/*genetics ; Huntington Disease/*genetics/*therapy ; Mice ; Mice, Knockout ; RNAi Therapeutics ; Trinucleotide Repeat Expansion/genetics ; },
}

Animals
CRISPR-Cas Systems/*genetics
Disease Models, Animal
*Gene Editing
Genetic Therapy/*methods
Humans
Huntingtin Protein/*genetics
Huntington Disease/*genetics/*therapy
Mice
Mice, Knockout
RNAi Therapeutics
Trinucleotide Repeat Expansion/genetics

@article {pmid29563188,
year = {2018},
author = {Li, M and Ouyang, H and Yuan, H and Li, J and Xie, Z and Wang, K and Yu, T and Liu, M and Chen, X and Tang, X and Jiao, H and Pang, D},
title = {Site-Specific Fat-1 Knock-In Enables Significant Decrease of n-6PUFAs/n-3PUFAs Ratio in Pigs.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {5},
pages = {1747-1754},
doi = {10.1534/g3.118.200114},
pmid = {29563188},
issn = {2160-1836},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/*genetics ; Fatty Acid Desaturases/*genetics ; Fatty Acids, Omega-3/*metabolism ; Fatty Acids, Omega-6/*metabolism ; Fetus/cytology ; Fibroblasts/metabolism ; *Gene Knock-In Techniques ; Genetic Loci ; Genetic Vectors/metabolism ; Genotype ; Mice ; *Mutagenesis, Site-Directed ; RNA, Untranslated/genetics ; Sus scrofa/*genetics ; Transcription, Genetic ; },
abstract = {The fat-1 gene from Caenorhabditis elegans encodes a fatty acid desaturase which was widely studied due to its beneficial function of converting n-6 polyunsaturated fatty acids (n-6PUFAs) to n-3 polyunsaturated fatty acids (n-3PUFAs). To date, many fat-1 transgenic animals have been generated to study disease pathogenesis or improve meat quality. However, all of them were generated using a random integration method with variable transgene expression levels and the introduction of selectable marker genes often raise biosafety concern. To this end, we aimed to generate marker-free fat-1 transgenic pigs in a site-specific manner. The Rosa26 locus, first found in mouse embryonic stem cells, has become one of the most common sites for inserting transgenes due to its safe and ubiquitous expression. In our study, the fat-1 gene was inserted into porcine Rosa 26 (pRosa26) locus via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. The Southern blot analysis of our knock-in pigs indicated a single copy of the fat-1 gene at the pRosa26 locus. Furthermore, this single-copy fat-1 gene supported satisfactory expression in a variety of tissues in F1 generation pigs. Importantly, the gas chromatography analysis indicated that these fat-1 knock-in pigs exhibited a significant increase in the level of n-3PUFAs, leading to an obvious decrease in the n-6PUFAs/n-3PUFAs ratio from 9.36 to 2.12 (***P < 0.0001). Altogether, our fat-1 knock-in pigs hold great promise for improving the nutritional value of pork and serving as an animal model to investigate therapeutic effects of n-3PUFAs on various diseases.},
}

Animals
Base Sequence
CRISPR-Cas Systems
Caenorhabditis elegans/*genetics
Caenorhabditis elegans Proteins/*genetics
Fatty Acid Desaturases/*genetics
Fatty Acids, Omega-3/*metabolism
Fatty Acids, Omega-6/*metabolism
Fetus/cytology
Fibroblasts/metabolism
*Gene Knock-In Techniques
Genetic Loci
Genetic Vectors/metabolism
Genotype
Mice
*Mutagenesis, Site-Directed
RNA, Untranslated/genetics
Sus scrofa/*genetics
Transcription, Genetic

@article {pmid29555822,
year = {2018},
author = {Li, Y and Ma, S and Sun, L and Zhang, T and Chang, J and Lu, W and Chen, X and Liu, Y and Wang, X and Shi, R and Zhao, P and Xia, Q},
title = {Programmable Single and Multiplex Base-Editing in Bombyx mori Using RNA-Guided Cytidine Deaminases.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {5},
pages = {1701-1709},
doi = {10.1534/g3.118.200134},
pmid = {29555822},
issn = {2160-1836},
mesh = {Animals ; Base Sequence ; Bombyx/enzymology/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Codon, Terminator/genetics ; Cytidine Deaminase/*genetics/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques ; Genes, Reporter ; Genome, Insect ; INDEL Mutation/genetics ; RNA, Guide/*genetics ; },
abstract = {Genome editing using standard tools (ZFN, TALEN, and CRISPR/Cas9) rely on double strand breaks to edit the genome. A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks was developed recently and quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites that can be edited by BE3 for inactivation, with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base editing in an invertebrate model.},
}

Animals
Base Sequence
Bombyx/enzymology/*genetics
CRISPR-Cas Systems/genetics
Cell Line
Codon, Terminator/genetics
Cytidine Deaminase/*genetics/metabolism
Gene Editing/*methods
Gene Knockout Techniques
Genes, Reporter
Genome, Insect
INDEL Mutation/genetics
RNA, Guide/*genetics

@article {pmid29548548,
year = {2018},
author = {Milligan, G and Inoue, A},
title = {Genome Editing Provides New Insights into Receptor-Controlled Signalling Pathways.},
journal = {Trends in pharmacological sciences},
volume = {39},
number = {5},
pages = {481-493},
doi = {10.1016/j.tips.2018.02.005},
pmid = {29548548},
issn = {1873-3735},
support = {BB/K019864/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L027887/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; GTP-Binding Protein alpha Subunits/genetics/metabolism ; *Gene Editing ; HEK293 Cells ; Humans ; Receptors, G-Protein-Coupled/*genetics/*metabolism ; Signal Transduction/genetics ; },
abstract = {Rapid developments in genome editing, based largely on CRISPR/Cas9 technologies, are offering unprecedented opportunities to eliminate the expression of single or multiple gene products in intact organisms and in model cell systems. Elimination of individual G protein-coupled receptors (GPCRs), both single and multiple G protein subunits, and arrestin adaptor proteins is providing new and sometimes unanticipated insights into molecular details of the regulation of cell signalling pathways and the behaviour of receptor ligands. Genome editing is certain to become a central component of therapeutic target validation, and will provide pharmacologists with new understanding of the complexities of action of novel and previously studied ligands, as well as of the transmission of signals from individual cell-surface receptors to intracellular signalling cascades.},
}

Animals
*CRISPR-Cas Systems
GTP-Binding Protein alpha Subunits/genetics/metabolism
*Gene Editing
HEK293 Cells
Humans
Receptors, G-Protein-Coupled/*genetics/*metabolism
Signal Transduction/genetics

@article {pmid29519936,
year = {2018},
author = {Katigbak, A and Robert, F and Paquet, M and Pelletier, J},
title = {Inducible Genome Editing with Conditional CRISPR/Cas9 Mice.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {5},
pages = {1627-1635},
doi = {10.1534/g3.117.300327},
pmid = {29519936},
issn = {2160-1836},
mesh = {Adoptive Transfer ; Animals ; CRISPR-Cas Systems/*genetics ; Doxycycline/pharmacology ; *Gene Editing ; Gene Expression/drug effects ; Genetic Engineering ; *Genome ; Hematopoietic Stem Cells/drug effects/metabolism ; Mice ; Mice, Transgenic ; Models, Animal ; Mouse Embryonic Stem Cells/drug effects/metabolism ; Proto-Oncogene Proteins c-myc/metabolism ; RNA, Guide/genetics ; },
abstract = {Genetically engineered mouse models (GEMMs) are powerful tools by which to probe gene function in vivo, obtain insight into disease etiology, and identify modifiers of drug response. Increased sophistication of GEMMs has led to the design of tissue-specific and inducible models in which genes of interest are expressed or ablated in defined tissues or cellular subtypes. Here we describe the generation of a transgenic mouse harboring a doxycycline-regulated Cas9 allele for inducible genome engineering. This model provides a flexible platform for genome engineering since editing is achieved by exogenous delivery of sgRNAs and should allow for the modeling of a range of biological and pathological processes.},
}

Adoptive Transfer
Animals
CRISPR-Cas Systems/*genetics
Doxycycline/pharmacology
*Gene Editing
Gene Expression/drug effects
Genetic Engineering
*Genome
Hematopoietic Stem Cells/drug effects/metabolism
Mice
Mice, Transgenic
Models, Animal
Mouse Embryonic Stem Cells/drug effects/metabolism
Proto-Oncogene Proteins c-myc/metabolism
RNA, Guide/genetics

@article {pmid29326075,
year = {2018},
author = {Ahmad, G and Amiji, M},
title = {Use of CRISPR/Cas9 gene-editing tools for developing models in drug discovery.},
journal = {Drug discovery today},
volume = {23},
number = {3},
pages = {519-533},
doi = {10.1016/j.drudis.2018.01.014},
pmid = {29326075},
issn = {1878-5832},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drug Discovery/methods ; Gene Editing/methods ; Genome/genetics ; Humans ; Models, Animal ; },
abstract = {Clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 (CRISPR/Cas9) enables targeted genome engineering. The simplicity of this system, its facile engineering, and amenability to multiplex genes make it the system of choice for many applications. This system has revolutionized our ability to carry out gene editing, transcription regulation, genome imaging, and epigenetic modification. In this review, we discuss the discovery of CRISPR/Cas9, its mechanism of action, its application in medicine and animal model development, and its delivery. We also highlight how the CRISPR/Cas9 system can affect the next generation of drugs by accelerating the identification and validation of high-value targets. The generation of precision disease models through this system will provide a rapid avenue for functional drug screening.},
}

Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Drug Discovery/methods
Gene Editing/methods
Genome/genetics
Humans
Models, Animal

@article {pmid28271030,
year = {2017},
author = {Ackermann, AM and Zhang, J and Heller, A and Briker, A and Kaestner, KH},
title = {High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting.},
journal = {Molecular metabolism},
volume = {6},
number = {3},
pages = {236-244},
doi = {10.1016/j.molmet.2017.01.003},
pmid = {28271030},
issn = {2212-8778},
support = {P30 DK019525/DK/NIDDK NIH HHS/United States ; P30 DK050306/DK/NIDDK NIH HHS/United States ; P30 DK019525/DK/NIDDK NIH HHS/United States ; P01 DK049210/DK/NIDDK NIH HHS/United States ; UC4 DK104119/DK/NIDDK NIH HHS/United States ; },
mesh = {3' Untranslated Regions/genetics ; Animals ; CRISPR-Cas Systems/drug effects ; Diabetes Mellitus, Type 2/genetics/metabolism ; Gene Knock-In Techniques ; Gene Targeting ; Genetic Engineering/*methods ; Genetic Techniques ; Glucagon/*genetics/metabolism ; Glucagon-Secreting Cells/metabolism/physiology ; Islets of Langerhans/drug effects ; Mice ; Mice, Transgenic/*genetics ; Tamoxifen/pharmacology ; Transgenes/drug effects ; },
}

3' Untranslated Regions/genetics
Animals
CRISPR-Cas Systems/drug effects
Diabetes Mellitus, Type 2/genetics/metabolism
Gene Knock-In Techniques
Gene Targeting
Genetic Engineering/*methods
Genetic Techniques
Glucagon/*genetics/metabolism
Glucagon-Secreting Cells/metabolism/physiology
Islets of Langerhans/drug effects
Mice
Mice, Transgenic/*genetics
Tamoxifen/pharmacology
Transgenes/drug effects

@article {pmid28266557,
year = {2017},
author = {Chen, J and Jiang, D and Tan, D and Fan, Z and Wei, Y and Li, M and Wang, D},
title = {Heterozygous mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in male tilapia, Oreochromis niloticus.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {43733},
doi = {10.1038/srep43733},
pmid = {28266557},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Order ; Gene Targeting ; *Genetic Association Studies ; Genetic Loci ; Gonads/metabolism ; *Heterozygote ; Infertility, Male/*genetics/metabolism ; Male ; *Mutation ; Peptide Elongation Factor 1/*genetics/metabolism ; Protein Transport ; Spermatogenesis/*genetics ; Tilapia/*genetics/metabolism ; Transcriptome ; },
abstract = {Eukaryotic elongation factor 1 alpha (eEF1A) is an essential component of the translational apparatus. In the present study, eEF1A1b was isolated from the Nile tilapia. Real-time PCR and Western blot revealed that eEF1A1b was expressed highly in the testis from 90 dah (days after hatching) onwards. In situ hybridization and immunohistochemistry analyses showed that eEF1A1b was highly expressed in the spermatogonia of the testis. CRISPR/Cas9 mediated mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in the F0 XY fish. Consistently, heterozygous mutation of eEF1A1b (eEF1A1b+/-) resulted in an absence of spermatocytes at 90 dah, very few spermatocytes, spermatids and spermatozoa at 180 dah, and decreased Cyp11b2 and serum 11-ketotestosterone level at both stages. Further examination of the fertilization capacity of the sperm indicated that the eEF1A1b+/- XY fish were infertile due to abnormal spermiogenesis. Transcriptomic analyses of the eEF1A1b+/- testis from 180 dah XY fish revealed that key elements involved in spermatogenesis, steroidogenesis and sperm motility were significantly down-regulated compared with the control XY. Transgenic overexpression of eEF1A1b rescued the spermatogenesis arrest phenotype of the eEF1A1b+/- testis. Taken together, our data suggested that eEF1A1b is crucial for spermatogenesis and male fertility in the Nile tilapia.},
}

Animals
CRISPR-Cas Systems
Gene Expression Profiling
Gene Expression Regulation
Gene Order
Gene Targeting
*Genetic Association Studies
Genetic Loci
Gonads/metabolism
*Heterozygote
Infertility, Male/*genetics/metabolism
Male
*Mutation
Peptide Elongation Factor 1/*genetics/metabolism
Protein Transport
Spermatogenesis/*genetics
Tilapia/*genetics/metabolism
Transcriptome

@article {pmid30388409,
year = {2018},
author = {Terns, MP},
title = {CRISPR-Based Technologies: Impact of RNA-Targeting Systems.},
journal = {Molecular cell},
volume = {72},
number = {3},
pages = {404-412},
doi = {10.1016/j.molcel.2018.09.018},
pmid = {30388409},
issn = {1097-4164},
abstract = {DNA-targeting CRISPR-Cas systems, such as those employing the RNA-guided Cas9 or Cas12 endonucleases, have revolutionized our ability to predictably edit genomes and control gene expression. Here, I summarize information on RNA-targeting CRISPR-Cas systems and describe recent advances in converting them into powerful and programmable RNA-binding and cleavage tools with a wide range of novel and important biotechnological and biomedical applications.},
}

@article {pmid30387552,
year = {2018},
author = {Kumlehn, J and Pietralla, J and Hensel, G and Pacher, M and Puchta, H},
title = {The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.12734},
pmid = {30387552},
issn = {1744-7909},
abstract = {Since the discovery that nucleases of the bacterial CRISPR/Cas system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double strand break induction, resulting in mutations by non-homologous recombination. Strategies for performing such experiments - from the design of guide RNA to the use of different transformation technologies - are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNA-binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.},
}

@article {pmid30386377,
year = {2018},
author = {Guzina, J and Chen, WH and Stankovic, T and Djordjevic, M and Zdobnov, E and Djordjevic, M},
title = {In silico Analysis Suggests Common Appearance of scaRNAs in Type II Systems and Their Association With Bacterial Virulence.},
journal = {Frontiers in genetics},
volume = {9},
number = {},
pages = {474},
doi = {10.3389/fgene.2018.00474},
pmid = {30386377},
issn = {1664-8021},
abstract = {In addition to its well-established defense function, CRISPR/Cas can also exhibit crucial non-canonical activity through endogenous gene expression regulation, which was found to mainly affect bacterial virulence. These non-canonical functions depend on scaRNA, which is a small RNA encoded outside of CRISPR array, that is typically flanked by a transcription start site (TSS) and a terminator, and is in part complementary to another small CRISPR/Cas-associated RNA (tracrRNAs). Identification of scaRNAs is however largely complicated by the scarcity of RNA-Seq data across different bacteria, so that they were identified only in a relatively rare CRISPR/Cas subtype (IIB), and the possibility of finding them in other Type II systems is currently unclear. This study presents the first effort toward systematic detection of small CRISPR/Cas-associated regulatory RNAs, where obtained predictions can guide future experiments. The core of our approach is ab initio detection of small RNAs from bacterial genome, which is based on jointly predicting transcription signals - TSS and terminators - and homology to CRISPR array repeat. Particularly, we employ our improved approach for detecting bacterial TSS, since accurate TSS detection is the main limiting factor for accurate small RNA prediction. We also explore how our predictions match to available RNA-Seq data and analyze their conservation across related bacterial species. In Type IIB systems, our predictions are consistent with experimental data, and we systematically identify scaRNAs throughout this subtype. Furthermore, we identify scaRNA:tracrRNA pairs in a number of IIA/IIC systems, where the appearance of scaRNAs co-occurs with the strains being pathogenic. RNA-Seq and conservation analysis show that our method is well suited for predicting CRISPR/Cas-associated small RNAs. We also find possible existence of a modified mechanism of CRISPR-associated small RNA action, which, interestingly, closely resembles the setup employed in biotechnological applications. Overall, our findings indicate that scaRNA:tracrRNA pairs are present in all subtypes of Type II systems, and point to an underlying connection with bacterial virulence. In addition to formulating these hypotheses, careful manual curation that we performed, makes an important first step toward fully automated predictor of CRISPR/Cas-associated small RNAs, which will allow their large scale analysis across diverse bacterial genomes.},
}

@article {pmid30386178,
year = {2018},
author = {Shankar, S and Sreekumar, A and Prasad, D and Das, AV and Pillai, MR},
title = {Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design.},
journal = {Cancer cell international},
volume = {18},
number = {},
pages = {169},
doi = {10.1186/s12935-018-0666-0},
pmid = {30386178},
issn = {1475-2867},
abstract = {Background: Gene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome.

Methods: HPV 16+ve cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole.

Results: Although ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity.

Conclusions: Our study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.},
}

@article {pmid30262503,
year = {2018},
author = {He, S and Del Viso, F and Chen, CY and Ikmi, A and Kroesen, AE and Gibson, MC},
title = {An axial Hox code controls tissue segmentation and body patterning in Nematostella vectensis.},
journal = {Science (New York, N.Y.)},
volume = {361},
number = {6409},
pages = {1377-1380},
doi = {10.1126/science.aar8384},
pmid = {30262503},
issn = {1095-9203},
mesh = {Animals ; Bacterial Proteins ; Body Patterning/*genetics ; CRISPR-Cas Systems ; Endoderm/cytology/growth & development ; Endonucleases ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques/methods ; Genes, Homeobox/genetics/*physiology ; Larva/cytology/genetics/growth & development ; Mutagenesis ; RNA, Small Interfering/genetics ; Sea Anemones/cytology/genetics/*growth & development ; Transcription Factors/genetics/*physiology ; },
abstract = {Hox genes encode conserved developmental transcription factors that govern anterior-posterior (A-P) pattering in diverse bilaterian animals, which display bilateral symmetry. Although Hox genes are also present within Cnidaria, these simple animals lack a definitive A-P axis, leaving it unclear how and when a functionally integrated Hox code arose during evolution. We used short hairpin RNA (shRNA)-mediated knockdown and CRISPR-Cas9 mutagenesis to demonstrate that a Hox-Gbx network controls radial segmentation of the larval endoderm during development of the sea anemone Nematostella vectensis. Loss of Hox-Gbx activity also elicits marked defects in tentacle patterning along the directive (orthogonal) axis of primary polyps. On the basis of our results, we propose that an axial Hox code may have controlled body patterning and tissue segmentation before the evolution of the bilaterian A-P axis.},
}

Animals
Bacterial Proteins
Body Patterning/*genetics
CRISPR-Cas Systems
Endoderm/cytology/growth & development
Endonucleases
*Gene Expression Regulation, Developmental
Gene Knockdown Techniques/methods
Genes, Homeobox/genetics/*physiology
Larva/cytology/genetics/growth & development
Mutagenesis
RNA, Small Interfering/genetics
Sea Anemones/cytology/genetics/*growth & development
Transcription Factors/genetics/*physiology

@article {pmid30167805,
year = {2018},
author = {Kroth, PG and Bones, AM and Daboussi, F and Ferrante, MI and Jaubert, M and Kolot, M and Nymark, M and Río Bártulos, C and Ritter, A and Russo, MT and Serif, M and Winge, P and Falciatore, A},
title = {Genome editing in diatoms: achievements and goals.},
journal = {Plant cell reports},
volume = {37},
number = {10},
pages = {1401-1408},
doi = {10.1007/s00299-018-2334-1},
pmid = {30167805},
issn = {1432-203X},
support = {GBMF4966//Gordon and Betty Moore Foundation/ ; },
mesh = {CRISPR-Cas Systems ; Diatoms/*genetics ; Gene Editing/*methods ; Genome ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; },
abstract = {Diatoms are major components of phytoplankton and play a key role in the ecology of aquatic ecosystems. These algae are of great scientific importance for a wide variety of research areas, ranging from marine ecology and oceanography to biotechnology. During the last 20 years, the availability of genomic information on selected diatom species and a substantial progress in genetic manipulation, strongly contributed to establishing diatoms as molecular model organisms for marine biology research. Recently, tailored TALEN endonucleases and the CRISPR/Cas9 system were utilized in diatoms, allowing targeted genetic modifications and the generation of knockout strains. These approaches are extremely valuable for diatom research because breeding, forward genetic screens by random insertion, and chemical mutagenesis are not applicable to the available model species Phaeodactylum tricornutum and Thalassiosira pseudonana, which do not cross sexually in the lab. Here, we provide an overview of the genetic toolbox that is currently available for performing stable genetic modifications in diatoms. We also discuss novel challenges that need to be addressed to fully exploit the potential of these technologies for the characterization of diatom biology and for metabolic engineering.},
}

CRISPR-Cas Systems
Diatoms/*genetics
Gene Editing/*methods
Genome
Transcription Activator-Like Effector Nucleases/genetics/metabolism

@article {pmid30074178,
year = {2018},
author = {Cheng, YX and Chen, GT and Yang, X and Wang, YQ and Hong, L},
title = {Effects of HPV Pseudotype Virus in Cutting E6 Gene Selectively in SiHa Cells.},
journal = {Current medical science},
volume = {38},
number = {2},
pages = {212-221},
doi = {10.1007/s11596-018-1868-3},
pmid = {30074178},
issn = {2523-899X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carcinogenesis/pathology ; Cell Line, Tumor ; Cell Proliferation ; Human papillomavirus 16/*metabolism ; Humans ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Oncogene Proteins, Viral/*genetics/metabolism ; Plasmids/metabolism ; RNA, Guide/metabolism ; RNA, Messenger/genetics/metabolism ; Repressor Proteins/*genetics/metabolism ; },
abstract = {The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer. After designing specific gRNA sequences targeting HPV16 E6, generating hCas9-EGFP and E6-gRNA-RFP plasmids, and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids, we determined the titer of the pseudotype virus using the TCID50 method. We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells. Experimental subjects were divided into control group, empty virus group, E6-gRNA transfected group, Cas9 transfected group and Cas9+E6-gRNA transfected group. The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis, and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups; the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups. Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6. The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells, and there were two cutting zones in the Cas9+E6-gRNA transfected group. However, the empty virus group, E6-gRNA transfected group and Cas9 transfected group had no corresponding zone. Compared with those in the control group, the empty virus group, E6-gRNA transfected group and Cas9 transfected group, the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9+E6-gRNA transfected group (P<0.01). In addition, the proliferation and migration abilities of SiHa cells were significantly inhibited (P<0.01). There were no significant differences among the other groups. In contrast to the control group, the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model (P<0.01). The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells, which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors. Taken together, these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease, particularly in HPV-related tumors.},
}

Animals
CRISPR-Cas Systems/genetics
Carcinogenesis/pathology
Cell Line, Tumor
Cell Proliferation
Human papillomavirus 16/*metabolism
Humans
Mice, Inbred BALB C
Mice, Nude
Neoplasm Invasiveness
Oncogene Proteins, Viral/*genetics/metabolism
Plasmids/metabolism
RNA, Guide/metabolism
RNA, Messenger/genetics/metabolism
Repressor Proteins/*genetics/metabolism

@article {pmid29511025,
year = {2018},
author = {Bertier, LD and Ron, M and Huo, H and Bradford, KJ and Britt, AB and Michelmore, RW},
title = {High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa).},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {5},
pages = {1513-1521},
doi = {10.1534/g3.117.300396},
pmid = {29511025},
issn = {2160-1836},
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Gene Knockout Techniques ; Genetic Markers ; Germ Cells/metabolism ; Germination/genetics ; Hot Temperature ; Inheritance Patterns/*genetics ; Lettuce/*genetics ; Mutation/genetics ; Phenotype ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; RNA, Guide/genetics ; Sequence Analysis, DNA ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature.},
}

Alleles
CRISPR-Cas Systems/*genetics
*Gene Editing
Gene Knockout Techniques
Genetic Markers
Germ Cells/metabolism
Germination/genetics
Hot Temperature
Inheritance Patterns/*genetics
Lettuce/*genetics
Mutation/genetics
Phenotype
Plant Proteins/*genetics/metabolism
Plants, Genetically Modified
RNA, Guide/genetics
Sequence Analysis, DNA
Transformation, Genetic

@article {pmid27436552,
year = {2016},
author = {Reid, W and O'Brochta, DA},
title = {Applications of genome editing in insects.},
journal = {Current opinion in insect science},
volume = {13},
number = {},
pages = {43-54},
doi = {10.1016/j.cois.2015.11.001},
pmid = {27436552},
issn = {2214-5753},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; *Gene Editing ; Genome, Insect/*genetics ; Insecta/*genetics ; },
abstract = {Insect genome editing was first reported 1991 in Drosophila melanogaster but the technology used was not portable to other species. Not until the recent development of facile, engineered DNA endonuclease systems has gene editing become widely available to insect scientists. Most applications in insects to date have been technical in nature but this is rapidly changing. Functional genomics and genetics-based insect control efforts will be major beneficiaries of the application of contemporary gene editing technologies. Engineered endonucleases like Cas9 make it possible to create powerful and effective gene drive systems that could be used to reduce or even eradicate specific insect populations. 'Best practices' for using Cas9-based editing are beginning to emerge making it easier and more effective to design and use but gene editing technologies still require traditional means of delivery in order to introduce them into somatic and germ cells of insects-microinjection of developing embryos. This constrains the use of these technologies by insect scientists. Insects created using editing technologies challenge existing governmental regulatory structures designed to manage genetically modified organisms.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems/genetics
Endonucleases/metabolism
*Gene Editing
Genome, Insect/*genetics
Insecta/*genetics

@article {pmid30382180,
year = {2018},
author = {Wang, HC and Wang, P and Chen, YW and Zhang, Y},
title = {Bevacizumab or fibronectin gene editing inhibits the osteoclastogenic effects of fibroblasts derived from human radicular cysts.},
journal = {Acta pharmacologica Sinica},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41401-018-0172-x},
pmid = {30382180},
issn = {1745-7254},
abstract = {Fibronectin (FN) is a main component of extracellular matrix (ECM) in most adult tissues. Under pathological conditions, particularly inflammation, wound healing and tumors, an alternatively spliced exon extra domain A (EDA) is included in the FN protein (EDA+FN), which facilitates cellular proliferation, motility, and aggressiveness in different lesions. In this study we investigated the effects of EDA+FN on bone destruction in human radicular cysts and explored the possibility of editing FN gene or blocking the related paracrine signaling pathway to inhibit the osteoclastogenesis. The specimens of radicular cysts were obtained from 20 patients. We showed that the vessel density was positively associated with both the lesion size (R = 0.49, P = 0.001) and EDA+FN staining (R = 0.26, P = 0.022) in the specimens. We isolated fibroblasts from surgical specimens, and used the CRISPR/Cas system to knockout the EDA exon, or used IST-9 antibody and bevacizumab to block EDA+FN and VEGF, respectively. Compared to control fibroblasts, the fibroblasts from radicular cysts exhibited significantly more Trap+MNCs, the relative expression level of VEGF was positively associated with both the ratio of EDA+FN/total FN (R = 0.271, P = 0.019) and with the number of Trap+MNCs (R = 0.331, P = 0.008). The knockout of the EDA exon significantly decreased VEGF expression in the fibroblasts derived from radicular cysts, leading to significantly decreased osteoclastogenesis; similar results were observed using bevacizumab to block VEGF, but block of EDA+FN with IST-9 antibody had no effect. Furthermore, the inhibitory effects of gene editing on Trap+MNC development were restored by exogenous VEGF. These results suggest that EDA+FN facilitates osteoclastogenesis in the fibrous capsule of radicular cysts, through a mechanism mediated by VEGF via an autocrine effect on the fibroblasts. Bevacizumab inhibits osteoclastogenesis in radicular cysts as effectively as the exclusion of the EDA exon by gene editing.},
}

@article {pmid29180469,
year = {2018},
author = {Yang, X and Wang, Z and Li, X and Liu, B and Liu, M and Liu, L and Chen, S and Ren, M and Wang, Y and Yu, M and Wang, B and Zou, J and Zhu, WG and Yin, Y and Gu, W and Luo, J},
title = {SHMT2 Desuccinylation by SIRT5 Drives Cancer Cell Proliferation.},
journal = {Cancer research},
volume = {78},
number = {2},
pages = {372-386},
doi = {10.1158/0008-5472.CAN-17-1912},
pmid = {29180469},
issn = {1538-7445},
mesh = {Animals ; Apoptosis ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems ; *Cell Proliferation ; Colonic Neoplasms/genetics/metabolism/*pathology ; Gene Expression Regulation, Neoplastic ; Glycine Hydroxymethyltransferase/antagonists & inhibitors/genetics/*metabolism ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; *Protein Processing, Post-Translational ; Serine/metabolism ; Sirtuins/genetics/*metabolism ; Succinates/chemistry/*metabolism ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {The mitochondrial serine hydroxymethyltransferase SHMT2, which catalyzes the rate-limiting step in serine catabolism, drives cancer cell proliferation, but how this role is regulated is undefined. Here, we report that the sirtuin SIRT5 desuccinylates SHMT2 to increase its activity and drive serine catabolism in tumor cells. SIRT5 interaction directly mediated desuccinylation of lysine 280 on SHMT2, which was crucial for activating its enzymatic activity. Conversely, hypersuccinylation of SHMT2 at lysine 280 was sufficient to inhibit its enzymatic activity and downregulate tumor cell growth in vitro and in vivo Notably, SIRT5 inactivation led to SHMT2 enzymatic downregulation and to abrogated cell growth under metabolic stress. Our results reveal that SHMT2 desuccinylation is a pivotal signal in cancer cells to adapt serine metabolic processes for rapid growth, and they highlight SIRT5 as a candidate target for suppressing serine catabolism as a strategy to block tumor growth.Significance: These findings reveal a novel mechanism for controlling cancer cell proliferation by blocking serine catabolism, as a general strategy to impede tumor growth. Cancer Res; 78(2); 372-86. ©2017 AACR.},
}

Animals
Apoptosis
Biomarkers, Tumor/genetics/*metabolism
CRISPR-Cas Systems
*Cell Proliferation
Colonic Neoplasms/genetics/metabolism/*pathology
Gene Expression Regulation, Neoplastic
Glycine Hydroxymethyltransferase/antagonists & inhibitors/genetics/*metabolism
Humans
Male
Mice
Mice, Inbred BALB C
Mice, Nude
*Protein Processing, Post-Translational
Serine/metabolism
Sirtuins/genetics/*metabolism
Succinates/chemistry/*metabolism
Tumor Cells, Cultured
Xenograft Model Antitumor Assays

@article {pmid29126790,
year = {2018},
author = {Jacob, P and Avni, A and Bendahmane, A},
title = {Translational Research: Exploring and Creating Genetic Diversity.},
journal = {Trends in plant science},
volume = {23},
number = {1},
pages = {42-52},
doi = {10.1016/j.tplants.2017.10.002},
pmid = {29126790},
issn = {1878-4372},
mesh = {CRISPR-Cas Systems ; Crops, Agricultural/*genetics ; Evolution, Molecular ; *Genetic Variation ; Plant Breeding/*methods ; Plants, Genetically Modified ; },
abstract = {The crop selection process has created a genetic bottleneck ultimately restricting breeding output. Wild relatives of major crops as well as the so-called 'neglected plant' species represent a reservoir of genetic diversity that remains underutilized. These species could be used as a tool to discover new alleles of agronomic interest or could be the target of breeding programs. Targeted induced local lesions in the genome (TILLING) can be used to translate in neglected crops what has been discovered in major crops and reciprocally. However, random mutagenesis, used in TILLING approaches, provides only a limited density of mutational events at a defined target locus. Alternatively, clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) fused to a cytidine deaminase could serve as a localized mutagenic agent to produce high-density mutant populations. Artificial evolution is at hand.},
}

CRISPR-Cas Systems
Crops, Agricultural/*genetics
Evolution, Molecular
*Genetic Variation
Plant Breeding/*methods
Plants, Genetically Modified

@article {pmid28256553,
year = {2017},
author = {Li, B and Cui, G and Shen, G and Zhan, Z and Huang, L and Chen, J and Qi, X},
title = {Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {43320},
doi = {10.1038/srep43320},
pmid = {28256553},
issn = {2045-2322},
mesh = {Agrobacterium/genetics/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Diterpenes/metabolism ; Diterpenes, Abietane ; Gene Editing/methods ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; *Genome, Plant ; Hydroxybenzoates/metabolism ; Ligases/deficiency/*genetics ; Mutagenesis ; Phenanthrenes ; Plant Proteins/*genetics/metabolism ; Plant Roots/*genetics/metabolism ; Plants, Medicinal ; Salvia miltiorrhiza/*genetics/metabolism ; Sequence Alignment ; },
abstract = {CRISPR/Cas9 is a powerful genome editing tool that has been extensively used in model plants and crops, such as Arabidopsis thaliana, rice, wheat, and soybean. Here, we report the use of CRISPR/Cas9 to precisely knock out the committed diterpene synthase gene (SmCPS1) involved in tanshinone biosynthesis in Salvia miltiorrhiza, a traditional Chinese medicinal herb with significant pharmacological activities, such as vasorelaxation, protection against ischemia-reperfusion injury, and antiarrhythmic effects. Three homozygous and eight chimeric mutants were obtained from 26 independent transgenic hairy root lines by Agrobacterium rhizogenes-mediated transformation. The metabolomic analysis based on LC-qTOF-MS and Q-TRAP-LC-MS/MS revealed that tanshinones, especially cryptotanshinone, tanshinone IIA and tanshinone I, are completely missing in homozygous mutants, without influencing other phenolic acid metabolites. By contrast, tanshinones are decreased but still detectable in chimeric mutants, which is similar to a previously-reported an RNAi study of SmCPS1. These results demonstrate that Agrobacterium rhizogenes- mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in S. miltiorrhiza, thus paving the way for large-scale genome editing in S. miltiorrhiza, which is important for pathway elucidation of secondary metabolites, quality improvement, and yield increases for this valuable traditional Chinese medicinal herb.},
}

Agrobacterium/genetics/metabolism
Base Sequence
*CRISPR-Cas Systems
Diterpenes/metabolism
Diterpenes, Abietane
Gene Editing/methods
Gene Expression Regulation, Plant
Gene Knockout Techniques
*Genome, Plant
Hydroxybenzoates/metabolism
Ligases/deficiency/*genetics
Mutagenesis
Phenanthrenes
Plant Proteins/*genetics/metabolism
Plant Roots/*genetics/metabolism
Plants, Medicinal
Salvia miltiorrhiza/*genetics/metabolism
Sequence Alignment

@article {pmid30375719,
year = {2018},
author = {Pickett, CJ and Zeller, RW},
title = {Efficient genome editing using CRISPR-Cas-mediated homology directed repair in the ascidian Ciona robusta.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {},
number = {},
pages = {},
doi = {10.1002/dvg.23260},
pmid = {30375719},
issn = {1526-968X},
abstract = {Eliminating or silencing a gene's level of activity is one of the classic approaches developmental biologists employ to determine a gene's function. A recently developed method of gene perturbation called CRISPR-Cas, which was derived from a prokaryotic adaptive immune system, has been adapted for use in eukaryotic cells. This technology has been established in several model organisms as a powerful and efficient tool for knocking out or knocking down the function of a gene of interest. It has been recently shown that CRISPR-Cas functions with fidelity and efficiency in Ciona robusta. Here, we show that in C. robusta CRISPR-Cas mediated genomic knock-ins can be efficiently generated. Electroporating a tissue-specific transgene driving Cas9 and a U6-driven gRNA transgene together with a fluorescent protein-containing homology directed repair (FP-HDR) template results in gene-specific patterns of fluorescence consistent with a targeted genomic insertion. Using the Tyrosinase locus to optimize reagents, we first characterize a new Pol III promoter for expressing gRNAs from the C. savignyi H1 gene, and then adapt technology that flanks gRNAs by ribozymes allowing cell-specific expression from Pol II promoters. Next, we examine homology arm-length efficiencies of FP-HDR templates. Reagents were then developed for targeting Brachyury and Pou4 that resulted in expected patterns of fluorescence, and sequenced PCR amplicons derived from single embryos validated predicted genomic insertions. Finally, using two differentially colored FP-HDR templates, we show that biallelic FP-HDR template insertion can be detected in live embryos of the F0 generation. This article is protected by copyright. All rights reserved.},
}

@article {pmid30374457,
year = {2018},
author = {England, WE and Kim, T and Whitaker, RJ},
title = {Metapopulation Structure of CRISPR-Cas Immunity in Pseudomonas aeruginosa and Its Viruses.},
journal = {mSystems},
volume = {3},
number = {5},
pages = {},
doi = {10.1128/mSystems.00075-18},
pmid = {30374457},
issn = {2379-5077},
abstract = {Viruses that infect the widespread opportunistic pathogen Pseudomonas aeruginosa have been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution of P. aeruginosa and its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark (R. L. Marvig, L. M. Sommer, S. Molin, and H. K. Johansen, Nat Genet 47:57-64, 2015, https://doi.org/10.1038/ng.3148). We show that new spacers are not added to or deleted from CRISPR arrays over time within a single patient but do vary among patients in this data set. We compared assembled CRISPR arrays from this data set to CRISPR arrays extracted from 726 additional publicly available P. aeruginosa sequences to show that local diversity in this population encompasses global diversity and that there is no evidence for population structure associated with location or environment sampled. We compare over 3,000 spacers from our global data set to 98 lytic and temperate viruses and proviruses and find a subset of related temperate virus clusters frequently targeted by CRISPR spacers. Highly targeted viruses are matched by different spacers in different arrays, resulting in a pattern of distributed immunity within the global population. Understanding the multiple immune contexts that P. aeruginosa viruses face can be applied to study of P. aeruginosa gene transfer, the spread of epidemic strains in cystic fibrosis patients, and viral control of P. aeruginosa infection. IMPORTANCE Pseudomonas aeruginosa is a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating how P. aeruginosa uses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicate P. aeruginosa and its viruses evolve in both a broad global population and in isolated human "islands." These data set the stage for exploring metapopulation dynamics occurring within and between isolated "island" populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains.},
}

@article {pmid30253789,
year = {2018},
author = {Gaj, T and Perez-Pinera, P},
title = {The continuously evolving CRISPR barcoding toolbox.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {143},
doi = {10.1186/s13059-018-1541-y},
pmid = {30253789},
issn = {1474-760X},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Two articles recently described the development of CRISPR technologies that have the potential to fundamentally transform the barcoding and tracing of mammalian cells.},
}

Animals
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid30093604,
year = {2018},
author = {Kalhor, R and Kalhor, K and Mejia, L and Leeper, K and Graveline, A and Mali, P and Church, GM},
title = {Developmental barcoding of whole mouse via homing CRISPR.},
journal = {Science (New York, N.Y.)},
volume = {361},
number = {6405},
pages = {},
doi = {10.1126/science.aat9804},
pmid = {30093604},
issn = {1095-9203},
support = {P50 HG005550/HG/NHGRI NIH HHS/United States ; R01 GM123313/GM/NIGMS NIH HHS/United States ; R01 MH103910/MH/NIMH NIH HHS/United States ; R01 CA222826/CA/NCI NIH HHS/United States ; R01 HG009285/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Cell Lineage ; Embryonic Development/*genetics ; Embryonic Stem Cells ; Gene Expression Profiling/*methods ; Mice ; Mutation ; RNA, Guide/genetics ; },
abstract = {In vivo barcoding using nuclease-induced mutations is a powerful approach for recording biological information, including developmental lineages; however, its application in mammalian systems has been limited. We present in vivo barcoding in the mouse with multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Activation upon conception and continued mutagenesis through gestation resulted in developmentally barcoded mice wherein information is recorded in lineage-specific mutations. We used these recordings for reliable post hoc reconstruction of the earliest lineages and investigation of axis development in the brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.},
}

Alleles
Animals
*CRISPR-Cas Systems
Cell Lineage
Embryonic Development/*genetics
Embryonic Stem Cells
Gene Expression Profiling/*methods
Mice
Mutation
RNA, Guide/genetics

@article {pmid29973285,
year = {2018},
author = {Tang, X and Liu, G and Zhou, J and Ren, Q and You, Q and Tian, L and Xin, X and Zhong, Z and Liu, B and Zheng, X and Zhang, D and Malzahn, A and Gong, Z and Qi, Y and Zhang, T and Zhang, Y},
title = {A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {84},
doi = {10.1186/s13059-018-1458-5},
pmid = {29973285},
issn = {1474-760X},
support = {31330017//National Natural Science Foundation of China (CN)/International ; 31771486//National Natural Science Foundation of China/International ; 2018ZX08022001-003//National Transgenic Major Project/International ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/*genetics ; Gene Editing/methods ; Genome, Plant/*genetics ; High-Throughput Nucleotide Sequencing/methods ; Mutation/genetics ; Oryza/*genetics ; RNA, Guide/genetics ; Whole Genome Sequencing/methods ; },
abstract = {BACKGROUND: Targeting specificity has been a barrier to applying genome editing systems in functional genomics, precise medicine and plant breeding. In plants, only limited studies have used whole-genome sequencing (WGS) to test off-target effects of Cas9. The cause of numerous discovered mutations is still controversial. Furthermore, WGS-based off-target analysis of Cpf1 (Cas12a) has not been reported in any higher organism to date.

RESULTS: We conduct a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice. The sequencing depths range from 45× to 105× with read mapping rates above 96%. Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and three Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation.

CONCLUSIONS: Our comprehensive and rigorous analysis of WGS data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Endonucleases/*genetics
Gene Editing/methods
Genome, Plant/*genetics
High-Throughput Nucleotide Sequencing/methods
Mutation/genetics
Oryza/*genetics
RNA, Guide/genetics
Whole Genome Sequencing/methods

@article {pmid29945655,
year = {2018},
author = {Chuai, G and Ma, H and Yan, J and Chen, M and Hong, N and Xue, D and Zhou, C and Zhu, C and Chen, K and Duan, B and Gu, F and Qu, S and Huang, D and Wei, J and Liu, Q},
title = {DeepCRISPR: optimized CRISPR guide RNA design by deep learning.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {80},
doi = {10.1186/s13059-018-1459-4},
pmid = {29945655},
issn = {1474-760X},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Computational Biology/methods ; Computer Simulation ; HCT116 Cells ; HEK293 Cells ; HL-60 Cells ; HeLa Cells ; Humans ; Machine Learning ; RNA Editing/genetics ; RNA, Guide/*genetics ; },
abstract = {A major challenge for effective application of CRISPR systems is to accurately predict the single guide RNA (sgRNA) on-target knockout efficacy and off-target profile, which would facilitate the optimized design of sgRNAs with high sensitivity and specificity. Here we present DeepCRISPR, a comprehensive computational platform to unify sgRNA on-target and off-target site prediction into one framework with deep learning, surpassing available state-of-the-art in silico tools. In addition, DeepCRISPR fully automates the identification of sequence and epigenetic features that may affect sgRNA knockout efficacy in a data-driven manner. DeepCRISPR is available at http://www.deepcrispr.net/ .},
}

CRISPR-Cas Systems/*genetics
Cell Line
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Computational Biology/methods
Computer Simulation
HCT116 Cells
HEK293 Cells
HL-60 Cells
HeLa Cells
Humans
Machine Learning
RNA Editing/genetics
RNA, Guide/*genetics

@article {pmid29845243,
year = {2018},
author = {Zhao, X and Zhou, H and Cheng, Y and Yu, W and Luo, G and Duan, C and Yao, F and Xiao, B and Feng, C and Xia, X and Wei, M and Wang, Y and Li, J and Dai, R},
title = {Reduction in activating transcription factor 4 promotes carbon tetrachloride and lipopolysaccharide/D‑galactosamine‑mediated liver injury in mice.},
journal = {Molecular medicine reports},
volume = {18},
number = {2},
pages = {1718-1725},
doi = {10.3892/mmr.2018.9080},
pmid = {29845243},
issn = {1791-3004},
mesh = {Activating Transcription Factor 4/*genetics/metabolism ; Alanine Transaminase/blood ; Animals ; Aspartate Aminotransferases/blood ; CRISPR-Cas Systems ; Carbon Tetrachloride/*administration & dosage ; Chemical and Drug Induced Liver Injury/*genetics/metabolism/pathology ; Endoplasmic Reticulum Stress/drug effects ; Eukaryotic Initiation Factor-2/genetics/metabolism ; Galactosamine/*administration & dosage ; Gene Editing ; Gene Expression Regulation ; JNK Mitogen-Activated Protein Kinases/genetics/metabolism ; Lipopolysaccharides/*administration & dosage ; Liver/drug effects/*metabolism/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Phosphorylation ; Plasmids/chemistry/metabolism ; Protective Factors ; Tunicamycin/pharmacology ; },
abstract = {Although activating transcription factor 4 (ATF4) is involved in the regulation of numerous biological functions, whether ATF4 has a direct role in liver injury is unknown. The aim of the present study was to investigate the role of ATF4 in liver injury using mouse models. The results revealed that ATF4 protein is expressed markedly higher in the mouse liver when in comparison with other tissues. Notably, tunicamycin treatment, an endoplasmic reticulum (ER) stress inducer, induced the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), but decreased ATF4 protein levels in the mouse liver. This suggested an unconventional regulation pattern of ATF4 protein not associated with ER stress or eIF2α. In addition, it was also observed that the liver levels of ATF4 protein were significantly reduced upon chronic liver injury induced by carbon tetrachloride (CCl4). ATF4 protein was also decreased in acute liver injury induced by lipopolysaccharide (LPS) plus D‑galactosamine (D‑GalN). Furthermore, the results revealed that knockdown of ATF4 by injecting ATF4‑targeting Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‑CRISPR associated protein 9 plasmids exacerbated CCl4 and LPS/D‑GalN‑induced liver injury as demonstrated by elevated serum aspartate transaminase and alanine aminotransferase levels. ATF4 suppression also enhanced CCl4 and LPS/D‑GalN mediated c‑Jun N‑terminal kinase activation. By contrast, ATF4 overexpression alleviated CCl4 and LPS/D‑GalN‑induced liver injury. Taken together, these observations suggested that ATF4 may serve a protective role in the mouse liver.},
}

Activating Transcription Factor 4/*genetics/metabolism
Alanine Transaminase/blood
Animals
Aspartate Aminotransferases/blood
CRISPR-Cas Systems
Carbon Tetrachloride/*administration & dosage
Chemical and Drug Induced Liver Injury/*genetics/metabolism/pathology
Endoplasmic Reticulum Stress/drug effects
Eukaryotic Initiation Factor-2/genetics/metabolism
Galactosamine/*administration & dosage
Gene Editing
Gene Expression Regulation
JNK Mitogen-Activated Protein Kinases/genetics/metabolism
Lipopolysaccharides/*administration & dosage
Liver/drug effects/*metabolism/pathology
Male
Mice
Mice, Inbred C57BL
Phosphorylation
Plasmids/chemistry/metabolism
Protective Factors
Tunicamycin/pharmacology

@article {pmid29657233,
year = {2018},
author = {Kaneko, T},
title = {Reproductive technologies for the generation and maintenance of valuable animal strains.},
journal = {The Journal of reproduction and development},
volume = {64},
number = {3},
pages = {209-215},
doi = {10.1262/jrd.2018-035},
pmid = {29657233},
issn = {1348-4400},
mesh = {Animals ; CRISPR-Cas Systems ; Electroporation/veterinary ; Gene Editing/*veterinary ; Gene Knockdown Techniques/*veterinary ; Genetic Engineering/methods ; Mice ; Organisms, Genetically Modified ; Rats ; Semen Preservation/*veterinary ; },
abstract = {Many types of mutant and genetically engineered strains have been produced in various animal species. Their numbers have dramatically increased in recent years, with new strains being rapidly produced using genome editing techniques. In the rat, it has been difficult to produce knockout and knock-in strains because the establishment of stem cells has been insufficient. However, a large number of knockout and knock-in strains can currently be produced using genome editing techniques, including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Microinjection technique has also contributed widely to the production of various kinds of genome edited animal strains. A novel electroporation method, the "Technique for Animal Knockout system by Electroporation (TAKE)" method, is a simple and highly efficient tool that has accelerated the production of new strains. Gamete preservation is extremely useful for maintaining large numbers of these valuable strains as genetic resources in the long term. These reproductive technologies, including microinjection, TAKE method, and gamete preservation, strongly support biomedical research and the bio-resource banking of animal models. In this review, we introduce the latest reproductive technologies used for the production of genetically engineered animals, especially rats, using genome editing techniques and the efficient maintenance of valuable strains as genetic resources. These technologies can also be applied to other laboratory animals, including mice, and domestic and wild animal species.},
}

Animals
CRISPR-Cas Systems
Electroporation/veterinary
Gene Editing/*veterinary
Gene Knockdown Techniques/*veterinary
Genetic Engineering/methods
Mice
Organisms, Genetically Modified
Rats
Semen Preservation/*veterinary

@article {pmid29657232,
year = {2018},
author = {Imaimatsu, K and Fujii, W and Hiramatsu, R and Miura, K and Kurohmaru, M and Kanai, Y},
title = {CRISPR/Cas9-mediated knock-in of the murine Y chromosomal Sry gene.},
journal = {The Journal of reproduction and development},
volume = {64},
number = {3},
pages = {283-287},
doi = {10.1262/jrd.2017-161},
pmid = {29657232},
issn = {1348-4400},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; *Genes, sry ; Male ; Mice ; Sex-Determining Region Y Protein/*genetics ; Testis/metabolism ; },
abstract = {Mammalian zygote-mediated genome editing via the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is widely used to generate genome-modified animals. This system allows for the production of loss-of-function mutations in various Y chromosome genes, including Sry, in mice. Here, we report the establishment of a CRISPR-Cas9-mediated knock-in line of Flag-tag sequences into the Sry locus at the C-terminal coding end of the Y chromosome (YSry-flag). In the F1 and successive generations, all male pups carrying the YSry-flag chromosome had normal testis differentiation and proper spermatogenesis at maturity, enabling complete fertility and the production of viable offspring. To our knowledge, this study is the first to produce a stable Sry knock-in line at the C-terminal region, highlighting a novel approach for examining the significance of amino acid changes at the naive Sry locus in mammals.},
}

Animals
*CRISPR-Cas Systems
Gene Editing
*Genes, sry
Male
Mice
Sex-Determining Region Y Protein/*genetics
Testis/metabolism

@article {pmid29423804,
year = {2018},
author = {Everman, JL and Rios, C and Seibold, MA},
title = {Primary Airway Epithelial Cell Gene Editing Using CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1706},
number = {},
pages = {267-292},
doi = {10.1007/978-1-4939-7471-9_15},
pmid = {29423804},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Epithelial Cells/*metabolism ; *Gene Deletion ; Gene Editing/*methods ; *Genome, Human ; Humans ; Respiratory Mucosa/*metabolism ; },
abstract = {The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction, allowing the CRISPR-Cas9 system to be integrated into the genome of the cell, resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel, advancements have been made in the culture, expansion, selection, and differentiation of AECs, which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods, we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery, (2) efficiently transduce AECs, (3) culture and select for a bulk edited AEC population, (4) molecularly screen AECs for Cas9 cutting and specific sequence edits, and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease.},
}

Animals
*CRISPR-Cas Systems
Epithelial Cells/*metabolism
*Gene Deletion
Gene Editing/*methods
*Genome, Human
Humans
Respiratory Mucosa/*metabolism

@article {pmid29321167,
year = {2018},
author = {Zhao, Y and Boeke, JD},
title = {Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {3},
pages = {789-796},
doi = {10.1534/g3.117.300363},
pmid = {29321167},
issn = {2160-1836},
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular ; Gene Order ; Gene Targeting/methods ; Genes, Fungal ; *Genetic Markers ; Genetic Vectors/*genetics ; Plasmids/genetics ; Schizosaccharomyces/*genetics ; *Sequence Deletion ; },
abstract = {Vectors encoding selectable markers have been widely used in yeast to maintain or express exogenous DNA fragments. In the fission yeast Schizosaccharomyces pombe, several engineered markers have been reported and widely used, such as ura4+ and ScLEU2 from Saccharomyces cerevisiae, which complement ura4 and leu1 mutations, respectively. These two auxotrophic markers share no homology with the S. pombe genome; however, most others can recombine with the genome due to sequence homology shared between the genomic and plasmid-borne copies of the markers. Here, we describe a CRISPR-Cas9 toolbox that can be used to quickly introduce "designer" auxotrophic marker deletions into host strains, including leu1-Δ0, his3-Δ0, and lys9-Δ0 Together with ura4-D18, this brings the total number of available designer deletion auxotrophic markers to four. The toolbox consists of a Cas9-gRNA expression vector and a donor DNA plasmid pair for each designer deletion. Using this toolbox, a set of auxotrophic S. pombe strains was constructed. Further, we reorganized essential components in the commonly used pREP series of plasmids and assembled the corresponding auxotrophic marker gene onto these plasmids. This toolbox for producing designer deletions, together with the newly developed strains and plasmids, will benefit the whole yeast community.},
}

*CRISPR-Cas Systems
Cloning, Molecular
Gene Order
Gene Targeting/methods
Genes, Fungal
*Genetic Markers
Genetic Vectors/*genetics
Plasmids/genetics
Schizosaccharomyces/*genetics
*Sequence Deletion

@article {pmid29295818,
year = {2018},
author = {Hu, P and Zhao, X and Zhang, Q and Li, W and Zu, Y},
title = {Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {3},
pages = {823-831},
doi = {10.1534/g3.117.300359},
pmid = {29295818},
issn = {2160-1836},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/*genetics ; *Gene Editing ; Gene Knockout Techniques ; Gene Targeting ; Genome ; Microinjections ; Mutagenesis ; Mutation ; *Nuclear Localization Signals ; Phenotype ; RNA, Guide ; *RNA, Messenger ; Recombinant Fusion Proteins ; Zebrafish/*genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites (tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system.},
}

Animals
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Endonucleases/*genetics
*Gene Editing
Gene Knockout Techniques
Gene Targeting
Genome
Microinjections
Mutagenesis
Mutation
*Nuclear Localization Signals
Phenotype
RNA, Guide
*RNA, Messenger
Recombinant Fusion Proteins
Zebrafish/*genetics

@article {pmid28211892,
year = {2017},
author = {Bian, S and Zhou, Y and Hu, Y and Cheng, J and Chen, X and Xu, Y and Liu, P},
title = {High-throughput in situ cell electroporation microsystem for parallel delivery of single guide RNAs into mammalian cells.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {42512},
doi = {10.1038/srep42512},
pmid = {28211892},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *Electroporation/instrumentation/methods ; Gene Editing ; Gene Expression ; *Gene Transfer Techniques ; Humans ; RNA, Guide/*genetics ; },
abstract = {Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells. Two plasmids encoding enhanced green and red fluorescent proteins (EGFP and ERFP), respectively, were successfully electroporated into attached HeLa cells on a 169-microwell array chip with transfection efficiencies of 71.6 ± 11.4% and 62.9 ± 2.7%, and a cell viability above 95%. We also successfully conducted selective electroporation of sgRNA into 293T cells expressing the Cas9 nuclease in a high-throughput manner and observed the four-fold increase of the GFP intensities due to the repair of the protein coding sequences mediated by the CRISPR/Cas9 system. This study proved that this HiCEP system has the great potential to be used for arrayed functional screens with genome-wide CRISPR libraries on hard-to-transfect cells in the future.},
}

Animals
CRISPR-Cas Systems
Cell Line
*Electroporation/instrumentation/methods
Gene Editing
Gene Expression
*Gene Transfer Techniques
Humans
RNA, Guide/*genetics

@article {pmid28209983,
year = {2017},
author = {Borgo, C and Franchin, C and Scalco, S and Bosello-Travain, V and Donella-Deana, A and Arrigoni, G and Salvi, M and Pinna, LA},
title = {Generation and quantitative proteomics analysis of CK2α/α'(-/-) cells.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {42409},
doi = {10.1038/srep42409},
pmid = {28209983},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Casein Kinase II/chemistry/genetics/*metabolism ; Catalysis ; Catalytic Domain ; Cell Line ; Chromatography, Liquid ; Gene Editing ; Gene Expression ; Gene Knockout Techniques ; Mice ; Protein Subunits ; Protein Transport ; *Proteome ; *Proteomics/methods ; Tandem Mass Spectrometry ; },
abstract = {CK2 is a ubiquitous, constitutively active, highly pleiotropic, acidophilic Ser/Thr protein kinase whose holoenzyme is composed of two catalytic (α and/or α') subunits and a dimer of a non-catalytic β subunit. Abnormally high CK2 level/activity is often associated with malignancy and a variety of cancer cells have been shown to rely on it to escape apoptosis. To gain information about the actual "druggability" of CK2 and to dissect CK2 dependent cellular processes that are instrumental to the establishment and progression of neoplasia we have exploited the CRISPR/Cas9 genome editing technology to generate viable clones of C2C12 myoblasts devoid of either both the CK2 catalytic subunits or its regulatory β-subunit. Suppression of both CK2 catalytic subunits promotes the disappearance of the β-subunit as well, through its accelerated proteasomal degradation. A quantitative proteomics analysis of CK2α/α'(-/-) versus wild type cells shows that knocking out both CK2 catalytic subunits causes a rearrangement of the proteomics profile, with substantially altered level (> 50%) of 240 proteins, 126 of which are up-regulated, while the other are down-regulated. A functional analysis reveals that up- and down-regulated proteins tend to be segregated into distinct sub-cellular compartments and play different biological roles, consistent with a global rewiring underwent by the cell to cope with the lack of CK2.},
}

Animals
CRISPR-Cas Systems
Casein Kinase II/chemistry/genetics/*metabolism
Catalysis
Catalytic Domain
Cell Line
Chromatography, Liquid
Gene Editing
Gene Expression
Gene Knockout Techniques
Mice
Protein Subunits
Protein Transport
*Proteome
*Proteomics/methods
Tandem Mass Spectrometry

@article {pmid28209967,
year = {2017},
author = {Raveux, A and Vandormael-Pournin, S and Cohen-Tannoudji, M},
title = {Optimization of the production of knock-in alleles by CRISPR/Cas9 microinjection into the mouse zygote.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {42661},
doi = {10.1038/srep42661},
pmid = {28209967},
issn = {2045-2322},
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Nucleus/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosol/metabolism ; Embryo, Mammalian ; Endonucleases/genetics/metabolism ; Exons ; Female ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Green Fluorescent Proteins/*genetics/metabolism ; Male ; Membrane Proteins/deficiency/*genetics ; Mice ; Mice, Transgenic ; Microinjections ; Mutation ; Oligodeoxyribonucleotides/genetics/metabolism ; Plasmids/administration & dosage/chemistry/*metabolism ; RNA, Guide/genetics/metabolism ; Zygote/cytology/metabolism ; },
abstract = {Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection. Our results also indicated that injection into the pronucleus compared to the cytoplasm is preferable to generate knock-in alleles with an oligonucleotide or a circular plasmid. Finally, we showed that Cas9D10A nickase variant was less efficient than wild-type Cas9 for generating knock-in alleles and caused a higher rate of mosaicism. Thus, our study provides valuable information that will help to improve the future production of precise genetic modifications in mice.},
}

Animals
Bacterial Proteins/genetics/metabolism
*CRISPR-Cas Systems
Cell Nucleus/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Cytosol/metabolism
Embryo, Mammalian
Endonucleases/genetics/metabolism
Exons
Female
Gene Editing/*methods
Gene Knock-In Techniques/*methods
Green Fluorescent Proteins/*genetics/metabolism
Male
Membrane Proteins/deficiency/*genetics
Mice
Mice, Transgenic
Microinjections
Mutation
Oligodeoxyribonucleotides/genetics/metabolism
Plasmids/administration & dosage/chemistry/*metabolism
RNA, Guide/genetics/metabolism
Zygote/cytology/metabolism

@article {pmid28198371,
year = {2017},
author = {Gong, H and Liu, M and Klomp, J and Merrill, BJ and Rehman, J and Malik, AB},
title = {Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {42127},
doi = {10.1038/srep42127},
pmid = {28198371},
issn = {2045-2322},
support = {R01 HL090152/HL/NHLBI NIH HHS/United States ; R01 HL125350/HL/NHLBI NIH HHS/United States ; T32 HL007829/HL/NHLBI NIH HHS/United States ; R01 GM094220/GM/NIGMS NIH HHS/United States ; R01 HL118068/HL/NHLBI NIH HHS/United States ; P01 HL060678/HL/NHLBI NIH HHS/United States ; },
mesh = {Adenoviridae/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Membrane Permeability ; Endonucleases/genetics/metabolism ; Endothelial Cells/cytology/drug effects/*metabolism ; Gene Editing/*methods ; Gene Expression ; Genes, Reporter ; Genetic Vectors/chemistry/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Lentivirus/*genetics/metabolism ; Lung/cytology/metabolism ; Mutagenesis, Site-Directed/*methods ; Mutation ; Primary Cell Culture ; RNA, Guide/genetics/metabolism ; Receptor, TIE-2/deficiency/*genetics ; Signal Transduction ; Thrombin/pharmacology ; },
abstract = {Human endothelial cells (ECs) are widely used to study mechanisms of angiogenesis, inflammation, and endothelial permeability. Targeted gene disruption induced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) nuclease gene editing is potentially an important tool for definitively establishing the functional roles of individual genes in ECs. We showed that co-delivery of adenovirus encoding EGFP-tagged Cas9 and lentivirus encoding a single guide RNA (sgRNA) in primary human lung microvascular ECs (HLMVECs) disrupted the expression of the Tie2 gene and protein. Tie2 disruption increased basal endothelial permeability and prevented permeability recovery following injury induced by the inflammatory stimulus thrombin. Thus, gene deletion via viral co-delivery of CRISPR-Cas9 in primary human ECs provides a novel platform to investigate signaling mechanisms of normal and perturbed EC function without the need for clonal expansion.},
}

Adenoviridae/*genetics/metabolism
*CRISPR-Cas Systems
Cell Membrane Permeability
Endonucleases/genetics/metabolism
Endothelial Cells/cytology/drug effects/*metabolism
Gene Editing/*methods
Gene Expression
Genes, Reporter
Genetic Vectors/chemistry/metabolism
Green Fluorescent Proteins/genetics/metabolism
High-Throughput Nucleotide Sequencing
Humans
Lentivirus/*genetics/metabolism
Lung/cytology/metabolism
Mutagenesis, Site-Directed/*methods
Mutation
Primary Cell Culture
RNA, Guide/genetics/metabolism
Receptor, TIE-2/deficiency/*genetics
Signal Transduction
Thrombin/pharmacology

@article {pmid28195201,
year = {2017},
author = {Hirose, M and Hasegawa, A and Mochida, K and Matoba, S and Hatanaka, Y and Inoue, K and Goto, T and Kaneda, H and Yamada, I and Furuse, T and Abe, K and Uenoyama, Y and Tsukamura, H and Wakana, S and Honda, A and Ogura, A},
title = {CRISPR/Cas9-mediated genome editing in wild-derived mice: generation of tamed wild-derived strains by mutation of the a (nonagouti) gene.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {42476},
doi = {10.1038/srep42476},
pmid = {28195201},
issn = {2045-2322},
mesh = {Agouti Signaling Protein/genetics ; Alleles ; Animals ; Animals, Wild ; *CRISPR-Cas Systems ; DNA Mutational Analysis ; Dopamine/metabolism ; *Gene Editing ; Gene Expression ; Humans ; Mesencephalon/metabolism ; Mice ; Mice, Knockout ; Mutation ; Phenotype ; },
abstract = {Wild-derived mice have contributed to experimental mouse genetics by virtue of their genetic diversity, which may help increase the chance of identifying novel modifier genes responsible for specific phenotypes and diseases. However, gene targeting using wild-derived mice has been unsuccessful because of the unavailability of stable embryonic stem cells. Here, we report that CRISPR/Cas9-mediated gene targeting can be applied to the Japanese wild-derived MSM/Ms strain (Mus musculus molossinus). We targeted the nonagouti (a) gene encoding the agouti protein that is localized in hair and the brain. We obtained three homozygous knockout mice as founders, all showing black coat colour. While homozygous knockout offspring were physiologically indistinguishable from wild-type litter-mates, they showed specific domesticated behaviours: hypoactivity in the dark phase and a decline in the avoidance of a human hand. These phenotypes were consistent over subsequent generations. Our findings support the empirical hypothesis that nonagouti is a domestication-linked gene, the loss of which might repress aggressive behaviour.},
}

Agouti Signaling Protein/genetics
Alleles
Animals
Animals, Wild
*CRISPR-Cas Systems
DNA Mutational Analysis
Dopamine/metabolism
*Gene Editing
Gene Expression
Humans
Mesencephalon/metabolism
Mice
Mice, Knockout
Mutation
Phenotype

@article {pmid30029480,
year = {2018},
author = {Bothwell, PJ and Kron, CD and Wittke, EF and Czerniak, BN and Bode, BP},
title = {Targeted Suppression and Knockout of ASCT2 or LAT1 in Epithelial and Mesenchymal Human Liver Cancer Cells Fail to Inhibit Growth.},
journal = {International journal of molecular sciences},
volume = {19},
number = {7},
pages = {},
doi = {10.3390/ijms19072093},
pmid = {30029480},
issn = {1422-0067},
mesh = {Amino Acid Transport System ASC/*metabolism ; Amino Acids/metabolism ; Biological Transport/drug effects ; CRISPR-Cas Systems/genetics ; Cell Death/drug effects ; Cell Proliferation/drug effects ; Epithelium/*pathology ; *Gene Knockout Techniques ; Humans ; Large Neutral Amino Acid-Transporter 1/*metabolism ; Liver Neoplasms/*metabolism/*pathology ; Mechanistic Target of Rapamycin Complex 1/metabolism ; Mesoderm/*pathology ; Mifepristone/pharmacology ; Minor Histocompatibility Antigens/*metabolism ; RNA, Antisense/metabolism ; RNA, Small Interfering/metabolism ; Signal Transduction/drug effects ; Sodium/metabolism ; },
abstract = {Amino acid transporters alanine-serine-cysteine transporter 2 (ASCT2) and L-Type Amino Acid Transporter 1 (LAT1) are coordinately enhanced in human cancers where among other roles, they are thought to drive mechanistic target-of-rapamycin (mTOR) growth signaling. To assess ASCT2 and LAT1 as therapeutic targets, nine unique short hairpin RNA (shRNA) vectors were used to stably suppress transporter expression in human epithelial (Hep3B) and mesenchymal (SK-Hep1) hepatocellular carcinoma (HCC) cell lines. In addition, six unique CRISPR-Cas9 vectors were used to edit the ASCT2 (SLC1A5) and LAT1 (SLC7A5) genes in epithelial (HUH7) and mesenchymal (SK-Hep1) HCC cells. Both approaches successfully diminished glutamine (ASCT2) and leucine (LAT1) initial-rate transport proportional to transporter protein suppression. In spite of profoundly reduced glutamine or leucine transport (up to 90%), transporter suppression or knockout failed to substantially affect cellular proliferation or basal and amino acid-stimulated mTORC1 growth signaling in either HCC cell type. Only LAT1 knockout in HUH7 slightly reduced growth rate. However, intracellular accumulation of radiolabeled glutamine and leucine over longer time periods largely recovered to control levels in ASCT2 and LAT1 knockout cells, respectively, which partially explains the lack of an impaired growth phenotype. These data collectively establish that in an in vitro context, human epithelial and mesenchymal HCC cell lines adapt to ASCT2 or LAT1 knockout. These results comport with an emerging model of amino acid exchangers like ASCT2 and LAT1 as "harmonizers", not drivers, of amino acid accumulation and signaling, despite their long-established dominant role in initial-rate amino acid transport.},
}

Amino Acid Transport System ASC/*metabolism
Amino Acids/metabolism
Biological Transport/drug effects
CRISPR-Cas Systems/genetics
Cell Death/drug effects
Cell Proliferation/drug effects
Epithelium/*pathology
*Gene Knockout Techniques
Humans
Large Neutral Amino Acid-Transporter 1/*metabolism
Liver Neoplasms/*metabolism/*pathology
Mechanistic Target of Rapamycin Complex 1/metabolism
Mesoderm/*pathology
Mifepristone/pharmacology
Minor Histocompatibility Antigens/*metabolism
RNA, Antisense/metabolism
RNA, Small Interfering/metabolism
Signal Transduction/drug effects
Sodium/metabolism

@article {pmid29901119,
year = {2018},
author = {Baliou, S and Adamaki, M and Kyriakopoulos, AM and Spandidos, DA and Panayiotidis, M and Christodoulou, I and Zoumpourlis, V},
title = {CRISPR therapeutic tools for complex genetic disorders and cancer (Review).},
journal = {International journal of oncology},
volume = {53},
number = {2},
pages = {443-468},
doi = {10.3892/ijo.2018.4434},
pmid = {29901119},
issn = {1791-2423},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Genetic Predisposition to Disease ; Genetic Therapy/*methods ; Humans ; Mice ; Neoplasms/genetics/*therapy ; Precision Medicine ; },
abstract = {One of the fundamental discoveries in the field of biology is the ability to modulate the genome and to monitor the functional outputs derived from genomic alterations. In order to unravel new therapeutic options, scientists had initially focused on inducing genetic alterations in primary cells, in established cancer cell lines and mouse models using either RNA interference or cDNA overexpression or various programmable nucleases [zinc finger nucleases (ZNF), transcription activator-like effector nucleases (TALEN)]. Even though a huge volume of data was produced, its use was neither cheap nor accurate. Therefore, the clustered regularly interspaced short palindromic repeats (CRISPR) system was evidenced to be the next step in genome engineering tools. CRISPR-associated protein 9 (Cas9)-mediated genetic perturbation is simple, precise and highly efficient, empowering researchers to apply this method to immortalized cancerous cell lines, primary cells derived from mouse and human origins, xenografts, induced pluripotent stem cells, organoid cultures, as well as the generation of genetically engineered animal models. In this review, we assess the development of the CRISPR system and its therapeutic applications to a wide range of complex diseases (particularly distinct tumors), aiming at personalized therapy. Special emphasis is given to organoids and CRISPR screens in the design of innovative therapeutic approaches. Overall, the CRISPR system is regarded as an eminent genome engineering tool in therapeutics. We envision a new era in cancer biology during which the CRISPR-based genome engineering toolbox will serve as the fundamental conduit between the bench and the bedside; nonetheless, certain obstacles need to be addressed, such as the eradication of side-effects, maximization of efficiency, the assurance of delivery and the elimination of immunogenicity.},
}

Animals
*CRISPR-Cas Systems
Cell Line, Tumor
Gene Editing
Genetic Predisposition to Disease
Genetic Therapy/*methods
Humans
Mice
Neoplasms/genetics/*therapy
Precision Medicine

@article {pmid29117363,
year = {2017},
author = {Jenks, S},
title = {New Gene-Editing Tool Slowly Advances Into the Clinic.},
journal = {Journal of the National Cancer Institute},
volume = {109},
number = {10},
pages = {},
doi = {10.1093/jnci/djx220},
pmid = {29117363},
issn = {1460-2105},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Metabolism, Inborn Errors/*genetics/*therapy ; },
}

*CRISPR-Cas Systems
Gene Editing/*methods
Genetic Therapy/*methods
Humans
Metabolism, Inborn Errors/*genetics/*therapy

@article {pmid28218310,
year = {2017},
author = {Lu, J and Shao, Y and Qin, X and Liu, D and Chen, A and Li, D and Liu, M and Wang, X},
title = {CRISPR knockout rat cytochrome P450 3A1/2 model for advancing drug metabolism and pharmacokinetics research.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {42922},
doi = {10.1038/srep42922},
pmid = {28218310},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cytochrome P-450 CYP3A/deficiency/*genetics ; Female ; Genotype ; Half-Life ; Intestine, Small/metabolism/pathology ; Liver/metabolism/pathology ; Male ; Microsomes, Liver/metabolism ; Nifedipine/analysis/metabolism/pharmacokinetics ; Phenotype ; Protein Isoforms/metabolism ; Rats ; Rats, Sprague-Dawley ; },
abstract = {Cytochrome P450 (CYP) 3A accounts for nearly 30% of the total CYP enzymes in the human liver and participates in the metabolism of over 50% of clinical drugs. Moreover, CYP3A plays an important role in chemical metabolism, toxicity, and carcinogenicity. New animal models are needed to investigate CYP3A functions, especially for drug metabolism. In this report, Cyp3a1/2 double knockout (KO) rats were generated by CRISPR-Cas9 technology, and then were characterized for viability and physiological status. The Cyp3a1/2 double KO rats were viable and fertile, and had no obvious physiological abnormities. Compared with the wild-type (WT) rat, Cyp3a1/2 expression was completely absent in the liver of the KO rat. In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats. The Cyp3a1/2 double KO rat model was successfully generated and characterized. The Cyp3a1/2 KO rats are a novel rodent animal model that will be a powerful tool for the study of the physiological and pharmacological roles of CYP3A, especially in drug and chemical metabolism in vivo.},
}

Animals
CRISPR-Cas Systems/*genetics
Cytochrome P-450 CYP3A/deficiency/*genetics
Female
Genotype
Half-Life
Intestine, Small/metabolism/pathology
Liver/metabolism/pathology
Male
Microsomes, Liver/metabolism
Nifedipine/analysis/metabolism/pharmacokinetics
Phenotype
Protein Isoforms/metabolism
Rats
Rats, Sprague-Dawley