@article {pmid30548045,
year = {2018},
author = {Tong, Y and Weber, T and Lee, SY},
title = {CRISPR/Cas-based genome engineering in natural product discovery.},
journal = {Natural product reports},
volume = {},
number = {},
pages = {},
doi = {10.1039/c8np00089a},
pmid = {30548045},
issn = {1460-4752},
abstract = {Covering: up to February, 2018This review briefly introduces and summarizes current knowledge about the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) - CRISPR/Cas system and how it was engineered to become one of the most important and versatile genome editing techniques that are currently revolutionizing the whole field of molecular biology. It aims to highlight and discuss the applications and remaining challenges of CRISPR/Cas (mainly focusing on CRISPR/SpCas9)-based genome editing in natural product discovery. The organisms covered include bacteria such as Streptomyces, Corynebacteria, and Myxobacteria; filamentous fungi such as Aspergillus, Beauveria, and Ganoderma; microalgae; and some plants. As closing remarks, the prospects of using CRISPR/Cas in natural product discovery will be discussed.},
}

@article {pmid30546095,
year = {2019},
author = {Fineran, PC},
title = {CRISPR-Cas impedes archaeal mating.},
journal = {Nature microbiology},
volume = {4},
number = {1},
pages = {2-3},
doi = {10.1038/s41564-018-0326-0},
pmid = {30546095},
issn = {2058-5276},
}

@article {pmid30544778,
year = {2018},
author = {Mayo-Muñoz, D and He, F and Jørgensen, JB and Madsen, PK and Bhoobalan-Chitty, Y and Peng, X},
title = {Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2.},
journal = {Viruses},
volume = {10},
number = {12},
pages = {},
doi = {10.3390/v10120695},
pmid = {30544778},
issn = {1999-4915},
support = {DFF-7017-00060//Danish Council for Independent Research/Technology and Production/ ; NNF17OC0031154//Novo Nordisk Foundation/Hallas-Møller Ascending Investigator Grant/ ; },
abstract = {Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) using the archaeon Sulfolobus islandicus LAL14/1 and its derivatives as hosts. The anti-CRISPR (Acr) gene acrID1, which inhibits CRISPR-Cas subtype I-D immunity, was first used as a selection marker to knock out genes from SIRV2M, an acrID1-null mutant of SIRV2. Moreover, we harnessed the endogenous CRISPR-Cas systems of the host to knock out the accessory genes consecutively, which resulted in a genome comprised solely of core genes of the 11 SIRV members. Furthermore, infection of this series of knockout mutants in the CRISPR-null host of LAL14/1 (Δarrays) confirmed the non-essentiality of the deleted genes and all except the last deletion mutant propagated as efficiently as the WT SIRV2. This suggested that the last gene deleted, SIRV2 gp37, is important for the efficient viral propagation. The generated viral mutants will be useful for future functional studies including searching for new Acrs and the approaches described in this case are applicable to other viruses.},
}

@article {pmid30523077,
year = {2018},
author = {Yan, WX and Hunnewell, P and Alfonse, LE and Carte, JM and Keston-Smith, E and Sothiselvam, S and Garrity, AJ and Chong, S and Makarova, KS and Koonin, EV and Cheng, DR and Scott, DA},
title = {Functionally diverse type V CRISPR-Cas systems.},
journal = {Science (New York, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1126/science.aav7271},
pmid = {30523077},
issn = {1095-9203},
abstract = {Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, g, h, and i. Cas12c, h, and i demonstrate RNA-guided double-stranded (ds) DNA interference activity. Cas12i exhibits markedly different efficiencies of crRNA spacer complementary and non-complementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided RNase with collateral RNase and single-stranded DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.},
}

@article {pmid30279181,
year = {2018},
author = {Jin, JJ and Lv, W and Xia, P and Xu, ZY and Zheng, AD and Wang, XJ and Wang, SS and Zeng, R and Luo, HM and Li, GL and Zuo, B},
title = {Long noncoding RNA SYISL regulates myogenesis by interacting with polycomb repressive complex 2.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {42},
pages = {E9802-E9811},
pmid = {30279181},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; *Epigenesis, Genetic ; Gene Silencing ; Mice ; Mice, Knockout ; Muscle Development/*physiology ; Polycomb Repressive Complex 2/genetics/*metabolism ; Promoter Regions, Genetic ; RNA, Long Noncoding/genetics/*metabolism ; },
abstract = {Although many long noncoding RNAs (lncRNAs) have been identified in muscle, their physiological function and regulatory mechanisms remain largely unexplored. In this study, we systematically characterized the expression profiles of lncRNAs during C2C12 myoblast differentiation and identified an intronic lncRNA, SYISL (SYNPO2 intron sense-overlapping lncRNA), that is highly expressed in muscle. Functionally, SYISL promotes myoblast proliferation and fusion but inhibits myogenic differentiation. SYISL knockout in mice results in significantly increased muscle fiber density and muscle mass. Mechanistically, SYISL recruits the enhancer of zeste homolog 2 (EZH2) protein, the core component of polycomb repressive complex 2 (PRC2), to the promoters of the cell-cycle inhibitor gene p21 and muscle-specific genes such as myogenin (MyoG), muscle creatine kinase (MCK), and myosin heavy chain 4 (Myh4), leading to H3K27 trimethylation and epigenetic silencing of target genes. Taken together, our results reveal that SYISL is a repressor of muscle development and plays a vital role in PRC2-mediated myogenesis.},
}

Animals
CRISPR-Cas Systems
Cell Differentiation
*Epigenesis, Genetic
Gene Silencing
Mice
Mice, Knockout
Muscle Development/*physiology
Polycomb Repressive Complex 2/genetics/*metabolism
Promoter Regions, Genetic
RNA, Long Noncoding/genetics/*metabolism

@article {pmid30201710,
year = {2018},
author = {Benej, M and Hong, X and Vibhute, S and Scott, S and Wu, J and Graves, E and Le, QT and Koong, AC and Giaccia, AJ and Yu, B and Chen, SC and Papandreou, I and Denko, NC},
title = {Papaverine and its derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {42},
pages = {10756-10761},
pmid = {30201710},
issn = {1091-6490},
support = {S10 OD020006/OD/NIH HHS/United States ; P01 CA067166/CA/NCI NIH HHS/United States ; R01 CA163581/CA/NCI NIH HHS/United States ; R15 CA202605/CA/NCI NIH HHS/United States ; P30 CA016058/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Hypoxia/*drug effects/radiation effects ; Cell Proliferation/drug effects/radiation effects ; Female ; Humans ; Lung Neoplasms/drug therapy/pathology/*radiotherapy ; Male ; Mice ; Mice, Inbred C57BL ; Mitochondria/*drug effects/metabolism/radiation effects ; NADH Dehydrogenase/*antagonists & inhibitors/genetics ; Oxygen/*metabolism ; Papaverine/*pharmacology ; Phosphodiesterase Inhibitors/pharmacology ; Radiation Tolerance ; Radiation-Sensitizing Agents/*pharmacology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {Tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. An acute increase in tumor oxygenation before radiation treatment should therefore significantly improve the tumor cell kill after radiation. Efforts to increase oxygen delivery to the tumor have not shown positive clinical results. Here we show that targeting mitochondrial respiration results in a significant reduction of the tumor cells' demand for oxygen, leading to increased tumor oxygenation and radiation response. We identified an activity of the FDA-approved drug papaverine as an inhibitor of mitochondrial complex I. We also provide genetic evidence that papaverine's complex I inhibition is directly responsible for increased oxygenation and enhanced radiation response. Furthermore, we describe derivatives of papaverine that have the potential to become clinical radiosensitizers with potentially fewer side effects. Importantly, this radiosensitizing strategy will not sensitize well-oxygenated normal tissue, thereby increasing the therapeutic index of radiotherapy.},
}

Animals
CRISPR-Cas Systems
Cell Hypoxia/*drug effects/radiation effects
Cell Proliferation/drug effects/radiation effects
Female
Humans
Lung Neoplasms/drug therapy/pathology/*radiotherapy
Male
Mice
Mice, Inbred C57BL
Mitochondria/*drug effects/metabolism/radiation effects
NADH Dehydrogenase/*antagonists & inhibitors/genetics
Oxygen/*metabolism
Papaverine/*pharmacology
Phosphodiesterase Inhibitors/pharmacology
Radiation Tolerance
Radiation-Sensitizing Agents/*pharmacology
Tumor Cells, Cultured
Xenograft Model Antitumor Assays

@article {pmid30018371,
year = {2018},
author = {Jeon, Y and Choi, YH and Jang, Y and Yu, J and Goo, J and Lee, G and Jeong, YK and Lee, SH and Kim, IS and Kim, JS and Jeong, C and Lee, S and Bae, S},
title = {Direct observation of DNA target searching and cleavage by CRISPR-Cas12a.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2777},
pmid = {30018371},
issn = {2041-1723},
mesh = {Acidaminococcus/enzymology/*genetics ; Base Pairing ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cloning, Molecular ; DNA/*genetics/metabolism ; DNA Cleavage ; Escherichia coli/enzymology/genetics ; Gene Editing ; Gene Expression ; Genetic Vectors/chemistry/metabolism ; Isoenzymes/genetics/metabolism ; Models, Molecular ; Nucleic Acid Conformation ; Nucleic Acid Heteroduplexes ; RNA, Guide/*genetics/metabolism ; Recombinant Proteins/genetics/metabolism ; },
abstract = {Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.},
}

Acidaminococcus/enzymology/*genetics
Base Pairing
Base Sequence
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
Cloning, Molecular
DNA/*genetics/metabolism
DNA Cleavage
Escherichia coli/enzymology/genetics
Gene Editing
Gene Expression
Genetic Vectors/chemistry/metabolism
Isoenzymes/genetics/metabolism
Models, Molecular
Nucleic Acid Conformation
Nucleic Acid Heteroduplexes
RNA, Guide/*genetics/metabolism
Recombinant Proteins/genetics/metabolism

@article {pmid30006570,
year = {2018},
author = {Liu, Z and Chen, M and Chen, S and Deng, J and Song, Y and Lai, L and Li, Z},
title = {Highly efficient RNA-guided base editing in rabbit.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2717},
pmid = {30006570},
issn = {2041-1723},
mesh = {Adenine/metabolism ; Adenosine Deaminase/genetics/metabolism ; Animals ; Base Pairing ; Base Sequence ; Blastocyst/cytology/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Codon, Nonsense ; Cytidine/metabolism ; Cytidine Deaminase/genetics/metabolism ; *Disease Models, Animal ; Founder Effect ; Gene Editing/*methods ; Humans ; INDEL Mutation ; Mutation, Missense ; RNA, Guide/*genetics/metabolism ; Rabbits/*genetics ; Sequence Alignment ; },
abstract = {Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53-88% and 44-100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit.},
}

Adenine/metabolism
Adenosine Deaminase/genetics/metabolism
Animals
Base Pairing
Base Sequence
Blastocyst/cytology/metabolism
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
Codon, Nonsense
Cytidine/metabolism
Cytidine Deaminase/genetics/metabolism
*Disease Models, Animal
Founder Effect
Gene Editing/*methods
Humans
INDEL Mutation
Mutation, Missense
RNA, Guide/*genetics/metabolism
Rabbits/*genetics
Sequence Alignment

@article {pmid29396264,
year = {2018},
author = {Gemberling, M and Gersbach, CA},
title = {Boosting, Not Breaking: CRISPR Activators Treat Disease Models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {2},
pages = {334-336},
pmid = {29396264},
issn = {1525-0024},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenomics ; *Transcriptional Activation ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Epigenomics
*Transcriptional Activation

@article {pmid29395380,
year = {2018},
author = {Schwartz, HT and Sternberg, PW},
title = {A Toolkit of Engineered Recombinational Balancers in C. elegans.},
journal = {Trends in genetics : TIG},
volume = {34},
number = {4},
pages = {253-255},
pmid = {29395380},
issn = {0168-9525},
support = {R24 OD023041/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Chromosomes ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Dejima and colleagues report using CRISPR/Cas9 to generate a new collection of greatly improved balancer chromosomes in the standard laboratory nematode Caenorhabditis elegans, using methods previously reported by the same laboratory, expanding the set of C. elegans balancers to cover nearly 90% of coding genes.},
}

Animals
*CRISPR-Cas Systems
Caenorhabditis elegans/*genetics
Chromosomes
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid29373314,
year = {2018},
author = {Tyurin-Kuzmin, PA and Karagyaur, MN and Rubtsov, YP and Dyikanov, DT and Vasiliev, PA and Vorotnikov, AV},
title = {CRISPR/Cas9-mediated modification of the extreme C-terminus impairs PDGF-stimulated activity of Duox2.},
journal = {Biological chemistry},
volume = {399},
number = {5},
pages = {437-446},
doi = {10.1515/hsz-2017-0229},
pmid = {29373314},
issn = {1437-4315},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Movement ; Cells, Cultured ; Dual Oxidases/genetics/*metabolism ; Fibroblasts/metabolism ; Mice ; Mutation ; NIH 3T3 Cells ; Platelet-Derived Growth Factor/*metabolism ; },
abstract = {Duox2 belongs to the large family of NADPH-oxidase enzymes that are implicated in immune response, vasoregulation, hormone synthesis, cell growth and differentiation via the regulated synthesis of H2O2 and reactive oxygen species. We and others have shown that Duox2 and H2O2 are involved in platelet-derived growth factor (PDGF) induced migration of fibroblasts. Now, using the CRISPR/Cas9-mediated genome editing we demonstrate that the extreme C-terminal region of Duox2 is required for PDGF-stimulated activity of Duox2 and H2O2 production. We generated the fibroblast cells that stably co-express the wild-type or C-terminally modified Duox2 and fluorescent H2O2 probe Hyper. We found that nonsense substitution of the last 23 amino acids in Duox2 results in complete loss of PDGF stimulation of intracellular H2O2 and fibroblast migration, yet these mutations have no effects on the expression of Duox2 and other NADPH-oxidases in cells. These findings illustrate for the first time that the extreme C-terminus of Duox2 is required for the functional activity of the enzyme. Furthermore, the conservative nature of the C-terminus suggests its role for activity in other NADPH-oxidases.},
}

Animals
CRISPR-Cas Systems/*genetics
Cell Movement
Cells, Cultured
Dual Oxidases/genetics/*metabolism
Fibroblasts/metabolism
Mice
Mutation
NIH 3T3 Cells
Platelet-Derived Growth Factor/*metabolism

@article {pmid28585549,
year = {2017},
author = {Rees, HA and Komor, AC and Yeh, WH and Caetano-Lopes, J and Warman, M and Edge, ASB and Liu, DR},
title = {Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15790},
pmid = {28585549},
issn = {2041-1723},
support = {R01 DC007174/DC/NIDCD NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R01 GM065400/GM/NIGMS NIH HHS/United States ; F32 GM112366/GM/NIGMS NIH HHS/United States ; R01 DC014089/DC/NIDCD NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; DNA/*genetics/metabolism ; Gene Editing ; Mice ; Protein Engineering ; Ribonucleoproteins/*genetics/metabolism ; Zebrafish ; },
abstract = {We recently developed base editing, a genome-editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here we describe two advances that address these limitations. First, we greatly reduce off-target base editing by installing mutations into our third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, we purify and deliver BE3 and HF-BE3 as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, we apply these advances to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo.},
}

Animals
CRISPR-Cas Systems
Cell Line
DNA/*genetics/metabolism
Gene Editing
Mice
Protein Engineering
Ribonucleoproteins/*genetics/metabolism
Zebrafish

@article {pmid28566761,
year = {2017},
author = {Sin, YY and Price, PR and Ballantyne, LL and Funk, CD},
title = {Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {2585},
pmid = {28566761},
issn = {2045-2322},
support = {U54 HD061221/HD/NICHD NIH HHS/United States ; MOP-341036//CIHR/Canada ; },
mesh = {Animals ; Arginase/*genetics/therapeutic use ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; *Gene Editing ; Genetic Therapy ; Hepatocytes/metabolism/pathology ; Humans ; Hyperargininemia/genetics/pathology/*therapy ; Induced Pluripotent Stem Cells/metabolism ; Liver/metabolism/pathology ; Mice ; Urea Cycle Disorders, Inborn/genetics/pathology/*therapy ; },
abstract = {Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro. While successful gene targeted repair was achieved, minimal urea cycle function was observed in the targeted iHLCs compared to adult hepatocytes likely due to inadequate maturation of the cells. On the other hand, iPSC-derived macrophages expressed substantial amounts of "repaired" arginase. Our studies provide proof-of-concept for gene-editing at the Arg1 locus and highlight the challenges that lie ahead to restore sufficient liver-based urea cycle function in patients with urea cycle disorders.},
}

Animals
Arginase/*genetics/therapeutic use
CRISPR-Cas Systems/genetics
Disease Models, Animal
*Gene Editing
Genetic Therapy
Hepatocytes/metabolism/pathology
Humans
Hyperargininemia/genetics/pathology/*therapy
Induced Pluripotent Stem Cells/metabolism
Liver/metabolism/pathology
Mice
Urea Cycle Disorders, Inborn/genetics/pathology/*therapy

@article {pmid30187755,
year = {2018},
author = {Yang, W and Restrepo-Pérez, L and Bengtson, M and Heerema, SJ and Birnie, A and van der Torre, J and Dekker, C},
title = {Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores.},
journal = {Nano letters},
volume = {18},
number = {10},
pages = {6469-6474},
pmid = {30187755},
issn = {1530-6992},
mesh = {Binding Sites ; Biosensing Techniques/*methods ; CRISPR-Cas Systems/*genetics ; DNA/chemistry/genetics/*isolation & purification ; Humans ; *Nanopores ; RNA, Guide/chemistry/genetics ; },
abstract = {Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.},
}

Binding Sites
Biosensing Techniques/*methods
CRISPR-Cas Systems/*genetics
DNA/chemistry/genetics/*isolation & purification
Humans
*Nanopores
RNA, Guide/chemistry/genetics

@article {pmid30013160,
year = {2018},
author = {Asada, S and Goyama, S and Inoue, D and Shikata, S and Takeda, R and Fukushima, T and Yonezawa, T and Fujino, T and Hayashi, Y and Kawabata, KC and Fukuyama, T and Tanaka, Y and Yokoyama, A and Yamazaki, S and Kozuka-Hata, H and Oyama, M and Kojima, S and Kawazu, M and Mano, H and Kitamura, T},
title = {Mutant ASXL1 cooperates with BAP1 to promote myeloid leukaemogenesis.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2733},
pmid = {30013160},
issn = {2041-1723},
mesh = {Animals ; Bone Marrow/metabolism/pathology ; Bone Marrow Transplantation ; CRISPR-Cas Systems ; Carcinogenesis/*genetics/metabolism/pathology ; Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; Female ; Gene Editing ; *Gene Expression Regulation, Leukemic ; HEK293 Cells ; HeLa Cells ; Homeodomain Proteins/genetics/metabolism ; Humans ; Interferon Regulatory Factors/genetics/metabolism ; Leukemia, Myeloid/*genetics/metabolism/mortality/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mutation ; RUNX1 Translocation Partner 1 Protein/genetics/metabolism ; Repressor Proteins/*genetics/metabolism ; Signal Transduction ; Survival Analysis ; Tumor Suppressor Proteins/*genetics/metabolism ; Ubiquitin Thiolesterase/*genetics/metabolism ; Ubiquitination ; Whole-Body Irradiation ; },
abstract = {ASXL1 mutations occur frequently in myeloid neoplasms and are associated with poor prognosis. However, the mechanisms by which mutant ASXL1 induces leukaemogenesis remain unclear. In this study, we report mutually reinforcing effects between a C-terminally truncated form of mutant ASXL1 (ASXL1-MT) and BAP1 in promoting myeloid leukaemogenesis. BAP1 expression results in increased monoubiquitination of ASXL1-MT, which in turn increases the catalytic function of BAP1. This hyperactive ASXL1-MT/BAP1 complex promotes aberrant myeloid differentiation of haematopoietic progenitor cells and accelerates RUNX1-ETO-driven leukaemogenesis. Mechanistically, this complex induces upregulation of posterior HOXA genes and IRF8 through removal of H2AK119 ubiquitination. Importantly, BAP1 depletion inhibits posterior HOXA gene expression and leukaemogenicity of ASXL1-MT-expressing myeloid leukemia cells. Furthermore, BAP1 is also required for the growth of MLL-fusion leukemia cells with posterior HOXA gene dysregulation. These data indicate that BAP1, which has long been considered a tumor suppressor, in fact plays tumor-promoting roles in myeloid neoplasms.},
}

Animals
Bone Marrow/metabolism/pathology
Bone Marrow Transplantation
CRISPR-Cas Systems
Carcinogenesis/*genetics/metabolism/pathology
Core Binding Factor Alpha 2 Subunit/genetics/metabolism
Female
Gene Editing
*Gene Expression Regulation, Leukemic
HEK293 Cells
HeLa Cells
Homeodomain Proteins/genetics/metabolism
Humans
Interferon Regulatory Factors/genetics/metabolism
Leukemia, Myeloid/*genetics/metabolism/mortality/pathology
Male
Mice
Mice, Inbred C57BL
Mutation
RUNX1 Translocation Partner 1 Protein/genetics/metabolism
Repressor Proteins/*genetics/metabolism
Signal Transduction
Survival Analysis
Tumor Suppressor Proteins/*genetics/metabolism
Ubiquitin Thiolesterase/*genetics/metabolism
Ubiquitination
Whole-Body Irradiation

@article {pmid29691708,
year = {2018},
author = {Cho, B and Kim, SJ and Lee, EJ and Ahn, SM and Lee, JS and Ji, DY and Lee, K and Kang, JT},
title = {Generation of insulin-deficient piglets by disrupting INS gene using CRISPR/Cas9 system.},
journal = {Transgenic research},
volume = {27},
number = {3},
pages = {289-300},
pmid = {29691708},
issn = {1573-9368},
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/*genetics ; Diabetes Mellitus/*genetics/pathology ; Embryo Transfer/methods ; Gene Knockout Techniques ; *Genetic Engineering ; Genotype ; Insulin/deficiency/*genetics ; Nuclear Transfer Techniques ; Phenotype ; Swine ; },
abstract = {Diabetes mellitus is a chronic disease with accompanying severe complications. Various animal models, mostly rodents due to availability of genetically modified lines, have been used to investigate the pathophysiology of diabetes. Using pigs for diabetic research can be beneficial because of their similarity in size, pathogenesis pathway, physiology, and metabolism with human. However, the use of pigs for diabetes research has been hampered due to only few pig models presenting diabetes symptoms. In this study, we have successfully generated insulin-deficient pigs by generating the indels of the porcine INS gene in somatic cells using CRISPR/Cas9 system followed by somatic cell nuclear transfer. First, somatic cells carrying a modified INS gene were generated using CRISPR/Cas9 system and their genotypes were confirmed by T7E1 assay; targeting efficiency was 40.4% (21/52). After embryo transfer, three live and five stillborn piglets were born. As expected, INS knockout piglets presented high blood glucose levels and glucose was detected in the urine. The level of insulin and c-peptide in the blood serum of INS knockout piglets were constant after feeding and the expression of insulin in the pancreas was absent in those piglets. This study demonstrates effectiveness of CRISPR/Cas9 system in generating novel pig models. We expect that these insulin-deficient pigs can be used in diabetes research to test the efficacy and safety of new drugs and the recipient of islet transplantation to investigate optimal transplantation strategies.},
}

Animals
Animals, Genetically Modified/genetics
CRISPR-Cas Systems/*genetics
Diabetes Mellitus/*genetics/pathology
Embryo Transfer/methods
Gene Knockout Techniques
*Genetic Engineering
Genotype
Insulin/deficiency/*genetics
Nuclear Transfer Techniques
Phenotype
Swine

@article {pmid29594927,
year = {2018},
author = {Lee, JH and Park, JH and Nam, TW and Seo, SM and Kim, JY and Lee, HK and Han, JH and Park, SY and Choi, YK and Lee, HW},
title = {Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout.},
journal = {Transgenic research},
volume = {27},
number = {3},
pages = {241-251},
pmid = {29594927},
issn = {1573-9368},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Gene Knockout Techniques/*methods ; Gene Targeting/methods ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Knockout/*genetics ; Mice, SCID/*genetics ; Phenotype ; T-Lymphocytes/immunology ; },
abstract = {Immunodeficient mice are widely used for pre-clinical studies to understand various human diseases. Here, we report the generation of four immunodeficient mouse models using CRISPR/Cas9 system without inserting any foreign gene sequences such as NeoR cassettes and their characterization. By eliminating any possible effects of adding a NeoR cassette, our mouse models may allow us to better elucidate the in vivo functions of each gene. Our FVB-Rag2-/-, B6-Rag2-/-, and BALB/c-Prkdc-/- mice showed phenotypes similar to those of the earlier immunodeficient mouse models, including a lack of mature B cells and T cells and an increase in the number of CD45+DX-5+ natural killer cells. However, B6-Il2rg-/- mice had a unique phenotype, with a lack of mature B cells, increased number of T cells, and decreased number of natural killer cells. Additionally, serum immunoglobulin levels in all four immunodeficient mouse models were significantly reduced when compared to those in wild-type mice with the exception of IgM in B6-Il2rg-/- mice. These results indicate that our immunodeficient mouse models are a robust tool for in vivo studies of the immune system and will provide new insights into the variation in phenotypic outcomes resulting from different gene-targeting methodologies.},
}

Animals
CRISPR-Cas Systems/*genetics
Disease Models, Animal
Gene Knockout Techniques/*methods
Gene Targeting/methods
Humans
Mice
Mice, Inbred BALB C
Mice, Knockout/*genetics
Mice, SCID/*genetics
Phenotype
T-Lymphocytes/immunology

@article {pmid29402189,
year = {2018},
author = {Kondrashov, A and Duc Hoang, M and Smith, JGW and Bhagwan, JR and Duncan, G and Mosqueira, D and Munoz, MB and Vo, NTN and Denning, C},
title = {Simplified Footprint-Free Cas9/CRISPR Editing of Cardiac-Associated Genes in Human Pluripotent Stem Cells.},
journal = {Stem cells and development},
volume = {27},
number = {6},
pages = {391-404},
pmid = {29402189},
issn = {1557-8534},
support = {RG/15/6/31436//British Heart Foundation/United Kingdom ; PG/09/027/27141//British Heart Foundation/United Kingdom ; NC/K000225/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research/International ; SP/15/9/31605//British Heart Foundation/United Kingdom ; PG/14/59/31000//British Heart Foundation/United Kingdom ; RG/14/1/30588//British Heart Foundation/United Kingdom ; MR/M017354/1//Medical Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Differentiation/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Humans ; Induced Pluripotent Stem Cells/*cytology ; Myocytes, Cardiac/*cytology ; },
abstract = {Modeling disease with human pluripotent stem cells (hPSCs) is hindered because the impact on cell phenotype from genetic variability between individuals can be greater than from the pathogenic mutation. While "footprint-free" Cas9/CRISPR editing solves this issue, existing approaches are inefficient or lengthy. In this study, a simplified PiggyBac strategy shortened hPSC editing by 2 weeks and required one round of clonal expansion and genotyping rather than two, with similar efficiencies to the longer conventional process. Success was shown across four cardiac-associated loci (ADRB2, GRK5, RYR2, and ACTC1) by genomic cleavage and editing efficiencies of 8%-93% and 8%-67%, respectively, including mono- and/or biallelic events. Pluripotency was retained, as was differentiation into high-purity cardiomyocytes (CMs; 88%-99%). Using the GRK5 isogenic lines as an exemplar, chronic stimulation with the β-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the β-blocker, propranolol. This shortened, footprint-free approach will be useful for mechanistic studies.},
}

CRISPR-Cas Systems/*genetics
Cell Differentiation/genetics
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/methods
Humans
Induced Pluripotent Stem Cells/*cytology
Myocytes, Cardiac/*cytology

@article {pmid29225034,
year = {2018},
author = {Weon, JL and Yang, SW and Potts, PR},
title = {Cytosolic Iron-Sulfur Assembly Is Evolutionarily Tuned by a Cancer-Amplified Ubiquitin Ligase.},
journal = {Molecular cell},
volume = {69},
number = {1},
pages = {113-125.e6},
doi = {10.1016/j.molcel.2017.11.010},
pmid = {29225034},
issn = {1097-4164},
mesh = {Animals ; CRISPR-Cas Systems ; Carrier Proteins/*metabolism ; Cell Line, Tumor ; DNA Damage/genetics ; DNA Repair/*genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Iron/*chemistry ; Iron-Sulfur Proteins/metabolism ; Lung Neoplasms/pathology ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Microtubule-Associated Proteins/genetics/*metabolism ; Neoplasm Proteins/genetics/*metabolism ; Sulfur/*chemistry ; Transcription Factors/*metabolism ; Ubiquitination ; },
abstract = {The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway functions to incorporate inorganic Fe-S cofactors into a variety of proteins, including several DNA repair enzymes. However, the mechanisms regulating the CIA pathway are unknown. We describe here that the MAGE-F1-NSE1 E3 ubiquitin ligase regulates the CIA pathway through ubiquitination and degradation of the CIA-targeting protein MMS19. Overexpression or knockout of MAGE-F1 altered Fe-S incorporation into MMS19-dependent DNA repair enzymes, DNA repair capacity, sensitivity to DNA-damaging agents, and iron homeostasis. Intriguingly, MAGE-F1 has undergone adaptive pseudogenization in select mammalian lineages. In contrast, MAGE-F1 is highly amplified in multiple human cancer types and amplified tumors have increased mutational burden. Thus, flux through the CIA pathway can be regulated by degradation of the substrate-specifying MMS19 protein and its downregulation is a common feature in cancer and is evolutionarily controlled.},
}

Animals
CRISPR-Cas Systems
Carrier Proteins/*metabolism
Cell Line, Tumor
DNA Damage/genetics
DNA Repair/*genetics
HEK293 Cells
HeLa Cells
Humans
Iron/*chemistry
Iron-Sulfur Proteins/metabolism
Lung Neoplasms/pathology
Male
Mice
Mice, Inbred NOD
Mice, SCID
Microtubule-Associated Proteins/genetics/*metabolism
Neoplasm Proteins/genetics/*metabolism
Sulfur/*chemistry
Transcription Factors/*metabolism
Ubiquitination

@article {pmid29031730,
year = {2017},
author = {Lundh, M and Pluciñska, K and Isidor, MS and Petersen, PSS and Emanuelli, B},
title = {Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA.},
journal = {Molecular metabolism},
volume = {6},
number = {10},
pages = {1313-1320},
pmid = {29031730},
issn = {2212-8778},
mesh = {Adipocytes/cytology/metabolism ; Adipocytes, Brown/metabolism ; Adipocytes, White/metabolism ; Adipogenesis/*genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Cell Differentiation/physiology ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression/genetics ; Gene Expression Regulation/genetics ; Genetic Engineering/*methods ; Mice ; RNA, Small Interfering/genetics/metabolism ; Thermogenesis ; Uncoupling Protein 1/metabolism ; },
abstract = {OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations.

METHODS: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes.

RESULTS: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations.

CONCLUSIONS: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.},
}

Adipocytes/cytology/metabolism
Adipocytes, Brown/metabolism
Adipocytes, White/metabolism
Adipogenesis/*genetics
Animals
CRISPR-Cas Systems/*genetics
Cell Differentiation/physiology
Cells, Cultured
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression/genetics
Gene Expression Regulation/genetics
Genetic Engineering/*methods
Mice
RNA, Small Interfering/genetics/metabolism
Thermogenesis
Uncoupling Protein 1/metabolism

@article {pmid28287154,
year = {2017},
author = {Chen, X and Lu, X and Shu, N and Wang, S and Wang, J and Wang, D and Guo, L and Ye, W},
title = {Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44304},
pmid = {28287154},
issn = {2045-2322},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Targeting/*methods ; Genome, Plant/genetics ; Gossypium/*genetics ; Models, Genetic ; *Mutagenesis ; Mutation ; Plant Proteins/genetics ; Plants, Genetically Modified ; Protoplasts/metabolism ; RNA, Guide/genetics ; Sequence Homology, Nucleic Acid ; },
abstract = {The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.},
}

Base Sequence
*CRISPR-Cas Systems
Gene Editing/*methods
Gene Targeting/*methods
Genome, Plant/genetics
Gossypium/*genetics
Models, Genetic
*Mutagenesis
Mutation
Plant Proteins/genetics
Plants, Genetically Modified
Protoplasts/metabolism
RNA, Guide/genetics
Sequence Homology, Nucleic Acid

@article {pmid30530241,
year = {2018},
author = {Goh, YJ and Barrangou, R},
title = {Harnessing CRISPR-Cas systems for precision engineering of designer probiotic lactobacilli.},
journal = {Current opinion in biotechnology},
volume = {56},
number = {},
pages = {163-171},
doi = {10.1016/j.copbio.2018.11.009},
pmid = {30530241},
issn = {1879-0429},
abstract = {Our evolving understanding on the mechanisms underlying the health-promoting attributes of probiotic lactobacilli, together with an expanding genome editing toolbox have made this genus an ideal chassis for the development of living therapeutics. The rising adoption of CRISPR-based technologies for prokaryotic engineering has demonstrated precise, efficient and scalable genome editing and tunable transcriptional regulation that can be translated into next-generation development of probiotic lactobacilli with enhanced robustness and designer functionalities. Here, we discuss how these tools in conjunction with the naturally abundant and diverse native CRISPR-Cas systems can be harnessed for Lactobacillus cell surface engineering and the delivery of biotherapeutics.},
}

@article {pmid30526490,
year = {2018},
author = {Li, X and Ma, Y and Liang, S and Tian, Y and Yin, S and Xie, S and Xie, H},
title = {Comparative genomics of 84 Pectobacterium genomes reveals the variations related to a pathogenic lifestyle.},
journal = {BMC genomics},
volume = {19},
number = {1},
pages = {889},
doi = {10.1186/s12864-018-5269-6},
pmid = {30526490},
issn = {1471-2164},
support = {BAIC07//Earmarked Fund for Beijing Innovation Consortium of Agriculture Research System/ ; },
abstract = {BACKGROUND: Pectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases of important crops and ornamental plants including soft rot, blackleg, and stem wilt. P. carotovorum is a genetically heterogeneous species consisting of three valid subspecies, P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), and P. carotovorum subsp. odoriferum (Pco).

RESULTS: Thirty-two P. carotovorum strains had their whole genomes sequenced, including the first complete genome of Pco and another circular genome of Pcb, as well as the high-coverage genome sequences for 30 additional strains covering Pcc, Pcb, and Pco. In combination with 52 other publicly available genome sequences, the comparative genomics study of P. carotovorum and other four closely related species P. polaris, P. parmentieri, P. atrosepticum, and Candidatus P. maceratum was conducted focusing on CRISPR-Cas defense systems and pathogenicity determinants. Our analysis identified two CRISPR-Cas types (I-F and I-E) in Pectobacterium, as well as another I-C type in Dickeya that is not found in Pectobacterium. The core pathogenicity factors (e.g., plant cell wall-degrading enzymes) were highly conserved, whereas some factors (e.g., flagellin, siderophores, polysaccharides, protein secretion systems, and regulatory factors) were varied among these species and/or subspecies. Notably, a novel type of T6SS as well as the sorbitol metabolizing srl operon was identified to be specific to Pco in Pectobacterium.

CONCLUSIONS: This study not only advances the available knowledge about the genetic differentiation of individual subspecies of P. carotovorum, but also delineates the general genetic features of P. carotovorum by comparison with its four closely related species, thereby substantially enriching the extent of information now available for functional genomic investigations about Pectobacterium.},
}

@article {pmid30526040,
year = {2018},
author = {Shabbir, MA and Wu, Q and Shabbir, MZ and Sajid, A and Ahmed, S and Sattar, A and Tang, Y and Li, J and Maan, MK and Hao, H and Yuan, Z},
title = {The CRISPR-cas system promotes antimicrobial resistance in Campylobacter jejuni.},
journal = {Future microbiology},
volume = {},
number = {},
pages = {},
doi = {10.2217/fmb-2018-0234},
pmid = {30526040},
issn = {1746-0921},
abstract = {AIM: The purpose of current study is to find out relationship between cas9 gene and antimicrobial resistance in C. jejuni NCTC11168.

MATERIALS & METHODS: The involvement of the cas9 gene in antimicrobial resistance of C. jejuni was determined by assessment of minimum inhibitory concentration, clustered regularly interspaced short palindromic repeats (CRISPR)-cas gene expression in standard strains, in vitro resistance development and transcriptome analysis of a cas9 deletion mutant and wild strains.

RESULTS: Increased expression of CRISPR-related genes was observed in standard strains. We also observed that Δcas9 mutant strain is more sensitive to antibiotics than its wild strain. Transcriptome analysis revealed that cas9 gene regulate several genes to promote antimicrobial resistance in C. jejuni.

CONCLUSION: CRISPR-cas system plays role in the enhancement of antimicrobial resistance in C. jejuni.},
}

@article {pmid30385564,
year = {2018},
author = {Kofler, N and Collins, JP and Kuzma, J and Marris, E and Esvelt, K and Nelson, MP and Newhouse, A and Rothschild, LJ and Vigliotti, VS and Semenov, M and Jacobsen, R and Dahlman, JE and Prince, S and Caccone, A and Brown, T and Schmitz, OJ},
title = {Editing nature: Local roots of global governance.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6414},
pages = {527-529},
doi = {10.1126/science.aat4612},
pmid = {30385564},
issn = {1095-9203},
mesh = {Animals ; Anthozoa/genetics ; *Bioethical Issues ; *CRISPR-Cas Systems ; Conservation of Natural Resources/*methods ; Coral Reefs ; Gene Editing/*ethics/*methods ; *Gene-Environment Interaction ; },
}

Animals
Anthozoa/genetics
*Bioethical Issues
*CRISPR-Cas Systems
Conservation of Natural Resources/*methods
Coral Reefs
Gene Editing/*ethics/*methods
*Gene-Environment Interaction

@article {pmid30309916,
year = {2018},
author = {Eldred, KC and Hadyniak, SE and Hussey, KA and Brenerman, B and Zhang, PW and Chamling, X and Sluch, VM and Welsbie, DS and Hattar, S and Taylor, J and Wahlin, K and Zack, DJ and Johnston, RJ},
title = {Thyroid hormone signaling specifies cone subtypes in human retinal organoids.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6411},
pages = {},
pmid = {30309916},
issn = {1095-9203},
support = {R01 EY025598/EY/NEI NIH HHS/United States ; T32 GM007231/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Embryonic Stem Cells/metabolism ; *Gene Expression Regulation, Developmental ; Humans ; Mutation ; Organoids/*growth & development/metabolism ; Proteolysis ; Retina/cytology/*growth & development ; Retinal Cone Photoreceptor Cells/*classification ; Thyroid Hormones/*metabolism ; },
abstract = {The mechanisms underlying specification of neuronal subtypes within the human nervous system are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity daytime and color vision. To determine the mechanism that controls S versus L/M fates, we studied the differentiation of human retinal organoids. Organoids and retinas have similar distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first, followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic expression of thyroid hormone-degrading and -activating proteins within the retina ensures low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair.},
}

CRISPR-Cas Systems
Cell Line
Embryonic Stem Cells/metabolism
*Gene Expression Regulation, Developmental
Humans
Mutation
Organoids/*growth & development/metabolism
Proteolysis
Retina/cytology/*growth & development
Retinal Cone Photoreceptor Cells/*classification
Thyroid Hormones/*metabolism

@article {pmid30293118,
year = {2018},
author = {Yang, X and Huang, B and Deng, L and Hu, Z},
title = {Progress in gene therapy using oncolytic vaccinia virus as vectors.},
journal = {Journal of cancer research and clinical oncology},
volume = {144},
number = {12},
pages = {2433-2440},
pmid = {30293118},
issn = {1432-1335},
mesh = {Animals ; CRISPR-Cas Systems ; Cancer Vaccines/genetics/immunology ; Gene Targeting ; *Genetic Therapy/methods ; Genetic Vectors/*genetics ; Humans ; Immunotherapy ; Neoplasms/genetics/immunology/therapy ; *Oncolytic Virotherapy/methods ; Oncolytic Viruses/*genetics ; Vaccinia virus/*genetics ; },
abstract = {BACKGROUND: Vaccinia virus was widely used in the World Health Organization's smallpox eradication campaign and is currently a promising vector for gene therapy owing to its unique characteristics. Vaccinia virus can selectively replicate and propagate productively in tumor cells, resulting in oncolysis. In addition, rapid viral particle production, wide host range, large genome size (approximately 200 kb), and safe handling render vaccinia virus a suitable vector for gene therapy.

MATERIALS AND METHODS: Cancer vaccines and gene therapy are being studied in clinical trials and experiment researches. However, we put forward unique challenges of optimal selection of foreign genes, administration and modification of VACV, personalized medicine, and other existing problems, based on current researches and our own experiments.

CONCLUSION: This review presents an overview of the vaccinia virus from its mechanisms to medical researches and clinical trials. We believe that the solution to these problems will contribute to understanding mechanisms of VACV and provide a theoretical basis for clinical treatment.},
}

Animals
CRISPR-Cas Systems
Cancer Vaccines/genetics/immunology
Gene Targeting
*Genetic Therapy/methods
Genetic Vectors/*genetics
Humans
Immunotherapy
Neoplasms/genetics/immunology/therapy
*Oncolytic Virotherapy/methods
Oncolytic Viruses/*genetics
Vaccinia virus/*genetics

@article {pmid30251692,
year = {2018},
author = {Luanpitpong, S and Poohadsuan, J and Samart, P and Kiratipaiboon, C and Rojanasakul, Y and Issaragrisil, S},
title = {Reactive oxygen species mediate cancer stem-like cells and determine bortezomib sensitivity via Mcl-1 and Zeb-1 in mantle cell lymphoma.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1864},
number = {11},
pages = {3739-3753},
doi = {10.1016/j.bbadis.2018.09.010},
pmid = {30251692},
issn = {1879-260X},
mesh = {Antineoplastic Agents/*pharmacology/therapeutic use ; Apoptosis/drug effects ; Biopsy ; Bortezomib/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Free Radical Scavengers/pharmacology ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Lymph Nodes/pathology ; Lymphoma, Mantle-Cell/*drug therapy/pathology ; Middle Aged ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors/genetics/*metabolism ; Neoplasm Recurrence, Local/pathology/prevention & control ; Neoplastic Stem Cells/drug effects/metabolism ; Primary Cell Culture ; Reactive Oxygen Species/*metabolism ; Zinc Finger E-box-Binding Homeobox 1/*metabolism ; },
abstract = {Mantle cell lymphoma (MCL) is an aggressive, incurable non-Hodgkin B-cell lymphoma with good initial response to therapy then subsequently relapse. Cancer stem cells (CSCs) are considered to be an underlying cause of these inevitable drug resistance and tumor regrowth, but how CSCs are regulated is largely unknown. We demonstrate here for the first time the existence of CSC-like subpopulations that are modulated by reactive oxygen species (ROS) in MCL cell lines and patient-derived primary cells in an inverse correlation with bortezomib (BTZ) sensitivity. Using various known donors and inhibitors of cellular superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH), we unveil their distinct roles in the regulation of CSC-like subpopulations and thus MCL response to BTZ. O2- inhibits CSC-like cells and sensitizes BTZ-induced apoptosis, whereas H2O2 conversely enriches CSC-like cells and protects against apoptosis and OH has minimal effects. We further observed that an anti-apoptotic Mcl-1 and a transcription factor Zeb-1 are favorable targets of O2- and H2O2, respectively. Using small molecule inhibition, ectopic expression and CRISPR/Cas9-mediated gene manipulation, we verified the roles of Mcl-1 and Zeb-1 in CSC and apoptosis regulation by O2- and H2O2. Our findings provide a novel mechanistic insight into the significance of redox status of MCL cells in determining their drug response via CSC-like subpopulations, which are imperative to a better understanding of therapeutic resistance and relapse.},
}

Antineoplastic Agents/*pharmacology/therapeutic use
Apoptosis/drug effects
Biopsy
Bortezomib/*pharmacology/therapeutic use
CRISPR-Cas Systems
Cell Line, Tumor
Cell Proliferation
Drug Resistance, Neoplasm
Female
Free Radical Scavengers/pharmacology
Gene Knockdown Techniques
HEK293 Cells
Humans
Lymph Nodes/pathology
Lymphoma, Mantle-Cell/*drug therapy/pathology
Middle Aged
Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors/genetics/*metabolism
Neoplasm Recurrence, Local/pathology/prevention & control
Neoplastic Stem Cells/drug effects/metabolism
Primary Cell Culture
Reactive Oxygen Species/*metabolism
Zinc Finger E-box-Binding Homeobox 1/*metabolism

@article {pmid30251682,
year = {2018},
author = {Montioli, R and Desbats, MA and Grottelli, S and Doimo, M and Bellezza, I and Borri Voltattorni, C and Salviati, L and Cellini, B},
title = {Molecular and cellular basis of ornithine δ-aminotransferase deficiency caused by the V332M mutation associated with gyrate atrophy of the choroid and retina.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1864},
number = {11},
pages = {3629-3638},
doi = {10.1016/j.bbadis.2018.08.032},
pmid = {30251682},
issn = {1879-260X},
mesh = {CRISPR-Cas Systems/genetics ; Coenzymes/metabolism ; Enzyme Assays ; Gene Knockout Techniques ; Gyrate Atrophy/drug therapy/*genetics/pathology ; HEK293 Cells ; Holoenzymes/genetics/metabolism ; Humans ; Mutagenesis, Site-Directed ; Ornithine-Oxo-Acid Transaminase/*genetics/metabolism ; Point Mutation ; Protein Aggregation, Pathological/drug therapy/*genetics/pathology ; Pyridoxal Phosphate/*metabolism ; Pyridoxine/pharmacology/therapeutic use ; Recombinant Proteins/genetics/metabolism ; Treatment Outcome ; Vitamin B Complex/*pharmacology/therapeutic use ; },
abstract = {Gyrate atrophy (GA) is a rare recessive disorder characterized by progressive blindness, chorioretinal degeneration and systemic hyperornithinemia. GA is caused by point mutations in the gene encoding ornithine δ-aminotransferase (OAT), a tetrameric pyridoxal 5'-phosphate-dependent enzyme catalysing the transamination of l-ornithine and α-ketoglutarate to glutamic-γ-semialdehyde and l-glutamate in mitochondria. More than 50 OAT variants have been identified, but their molecular and cellular properties are mostly unknown. A subset of patients is responsive to pyridoxine administration, although the mechanisms underlying responsiveness have not been clarified. Herein, we studied the effects of the V332M mutation identified in pyridoxine-responsive patients. The Val332-to-Met substitution does not significantly affect the spectroscopic and kinetic properties of OAT, but during catalysis it makes the protein prone to convert into the apo-form, which undergoes unfolding and aggregation under physiological conditions. By using the CRISPR/Cas9 technology we generated a new cellular model of GA based on HEK293 cells knock-out for the OAT gene (HEK-OAT_KO). When overexpressed in HEK-OAT_KO cells, the V332M variant is present in an inactive apodimeric form, but partly shifts to the catalytically-competent holotetrameric form in the presence of exogenous PLP, thus explaining the responsiveness of these patients to pyridoxine administration. Overall, our data represent the first integrated molecular and cellular analysis of the effects of a pathogenic mutation in OAT. In addition, we validated a novel cellular model for the disease that could prove instrumental to define the molecular defect of other GA-causing variants, as well as their responsiveness to pyridoxine and other putative drugs.},
}

CRISPR-Cas Systems/genetics
Coenzymes/metabolism
Enzyme Assays
Gene Knockout Techniques
Gyrate Atrophy/drug therapy/*genetics/pathology
HEK293 Cells
Holoenzymes/genetics/metabolism
Humans
Mutagenesis, Site-Directed
Ornithine-Oxo-Acid Transaminase/*genetics/metabolism
Point Mutation
Protein Aggregation, Pathological/drug therapy/*genetics/pathology
Pyridoxal Phosphate/*metabolism
Pyridoxine/pharmacology/therapeutic use
Recombinant Proteins/genetics/metabolism
Treatment Outcome
Vitamin B Complex/*pharmacology/therapeutic use

@article {pmid30241032,
year = {2018},
author = {Han, YQ and Ming, SL and Wu, HT and Zeng, L and Ba, G and Li, J and Lu, WF and Han, J and Du, QJ and Sun, MM and Yang, GY and Wang, J and Chu, BB},
title = {Myostatin knockout induces apoptosis in human cervical cancer cells via elevated reactive oxygen species generation.},
journal = {Redox biology},
volume = {19},
number = {},
pages = {412-428},
pmid = {30241032},
issn = {2213-2317},
mesh = {A549 Cells ; Animals ; Antioxidants/pharmacology ; Apoptosis/*genetics ; CRISPR-Cas Systems/genetics ; Cachexia/*pathology ; Caspases/metabolism ; Cell Line, Tumor ; Cell Proliferation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytochromes c/metabolism ; Epoxy Compounds/pharmacology ; Fatty Acids/metabolism ; Female ; Gene Knockout Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Lipid Metabolism/physiology ; Membrane Potential, Mitochondrial/genetics ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mitochondria/genetics/metabolism ; Myostatin/*genetics ; Oxidation-Reduction ; Reactive Oxygen Species/*metabolism ; Uterine Cervical Neoplasms/genetics/*pathology ; Xenograft Model Antitumor Assays ; },
abstract = {Myostatin (Mstn) is postulated to be a key determinant of muscle loss and cachexia in cancer. However, no experimental evidence supports a role for Mstn in cancer, particularly in regulating the survival and growth of cancer cells. In this study, we showed that the expression of Mstn was significantly increased in different tumor tissues and human cancer cells. Mstn knockdown inhibited the proliferation of cancer cells. A knockout (KO) of Mstn created by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 (CRISPR/Cas9) induced mitochondria-dependent apoptosis in HeLa cells. Furthermore, KO of Mstn reduced the lipid content. Molecular analyses demonstrated that the expression levels of fatty acid oxidation-related genes were upregulated and then increased rate of fatty acid oxidation. Mstn deficiency-induced apoptosis took place along with generation of reactive oxygen species (ROS) and elevated fatty acid oxidation, which may play a role in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, apoptosis induced by Mstn KO was partially rescued by antioxidants and etomoxir, thereby suggesting that the increased level of ROS was functionally involved in mediating apoptosis. Overall, our findings demonstrate a novel function of Mstn in regulating mitochondrial metabolism and apoptosis within cancer cells. Hence, inhibiting the production and function of Mstn may be an effective therapeutic intervention during cancer progression and muscle loss in cachexia.},
}

A549 Cells
Animals
Antioxidants/pharmacology
Apoptosis/*genetics
CRISPR-Cas Systems/genetics
Cachexia/*pathology
Caspases/metabolism
Cell Line, Tumor
Cell Proliferation/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cytochromes c/metabolism
Epoxy Compounds/pharmacology
Fatty Acids/metabolism
Female
Gene Knockout Techniques
HEK293 Cells
HeLa Cells
Humans
Lipid Metabolism/physiology
Membrane Potential, Mitochondrial/genetics
Mice
Mice, Inbred BALB C
Mice, Nude
Mitochondria/genetics/metabolism
Myostatin/*genetics
Oxidation-Reduction
Reactive Oxygen Species/*metabolism
Uterine Cervical Neoplasms/genetics/*pathology
Xenograft Model Antitumor Assays

@article {pmid30222157,
year = {2018},
author = {Sorlien, EL and Witucki, MA and Ogas, J},
title = {Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {138},
pages = {},
pmid = {30222157},
issn = {1940-087X},
support = {P30 CA023168/CA/NCI NIH HHS/United States ; R21 CA182197/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Knockout Techniques/*methods ; Zebrafish/*genetics ; },
abstract = {Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.},
}

Animals
CRISPR-Cas Systems/*genetics
Gene Knockout Techniques/*methods
Zebrafish/*genetics

@article {pmid30219987,
year = {2018},
author = {Hu, L and Zhang, H and Yang, Q and Meng, Q and Han, S and Nwafor, CC and Khan, MHU and Fan, C and Zhou, Y},
title = {Promoter variations in a homeobox gene, BnA10.LMI1, determine lobed leaves in rapeseed (Brassica napus L.).},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {131},
number = {12},
pages = {2699-2708},
pmid = {30219987},
issn = {1432-2242},
support = {2015CB150202//National Key Basic Research Program of China/ ; 2662015PY172//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Alleles ; Amino Acid Sequence ; Brassica napus/anatomy & histology/*genetics ; CRISPR-Cas Systems ; Chromosome Mapping ; *Genes, Homeobox ; *Genes, Plant ; Genetic Linkage ; Plant Leaves/*anatomy & histology ; Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; *Promoter Regions, Genetic ; Transcription Factors/genetics ; },
abstract = {KEY MESSAGE: BnA10.LMI1 positively regulates the development of leaf lobes in Brassica napus, and cis-regulatory divergences cause the different allele effects. Leaf shape is an important agronomic trait, and large variations in this trait exist within the Brassica germplasm. The lobed leaf is a unique morphological characteristic for Brassica improvement. Nevertheless, the molecular basis of leaf lobing in Brassica is poorly understood. Here, we show that an incompletely dominant locus, BnLLA10, is responsible for the lobed-leaf shape in rapeseed. A LATE MERISTEM IDENTITY1 (LMI1)-like gene (BnA10.LMI1) encoding an HD-Zip I transcription factor is the causal gene underlying the BnLLA10 locus. Sequence analysis of parental alleles revealed no sequence variations in the coding sequences, whereas abundant variations were identified in the regulatory region. Consistent with this finding, the expression levels of BnLMI1 were substantially elevated in the lobed-leaf parent compared with its near-isogenic line. The knockout mutations of BnA10.LMI1 gene were induced using the CRISPR/Cas9 system in both HY (the lobed-leaf parent) and J9707 (serrated leaf) genetic backgrounds. BnA10.LMI1 null mutations in the HY background were sufficient to produce unlobed leaves, whereas null mutations in the J9707 background showed no obvious changes in leaf shape compared with the control. Collectively, our results indicate that BnA10.LMI1 positively regulates the development of leaf lobes in B. napus, with cis-regulatory divergences causing the different allelic effects, providing new insights into the molecular mechanism of leaf lobe formation in Brassica crops.},
}

Alleles
Amino Acid Sequence
Brassica napus/anatomy & histology/*genetics
CRISPR-Cas Systems
Chromosome Mapping
*Genes, Homeobox
*Genes, Plant
Genetic Linkage
Plant Leaves/*anatomy & histology
Plant Proteins/genetics
Plants, Genetically Modified/genetics
*Promoter Regions, Genetic
Transcription Factors/genetics

@article {pmid30199041,
year = {2018},
author = {Haupt, A and Grancharova, T and Arakaki, J and Fuqua, MA and Roberts, B and Gunawardane, RN},
title = {Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {138},
pages = {},
pmid = {30199041},
issn = {1940-087X},
mesh = {CRISPR-Cas Systems/*immunology ; Cells, Cultured ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; },
abstract = {A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.},
}

CRISPR-Cas Systems/*immunology
Cells, Cultured
Humans
Induced Pluripotent Stem Cells/*metabolism

@article {pmid30190307,
year = {2018},
author = {Watters, KE and Fellmann, C and Bai, HB and Ren, SM and Doudna, JA},
title = {Systematic discovery of natural CRISPR-Cas12a inhibitors.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6411},
pages = {236-239},
pmid = {30190307},
issn = {1095-9203},
support = {K99 GM118909/GM/NIGMS NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/*antagonists & inhibitors/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Computational Biology/methods ; DNA Cleavage ; Endonucleases/*antagonists & inhibitors ; *Gene Editing ; Genome, Bacterial ; Humans ; Moraxella/*genetics ; },
abstract = {Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.},
}

Bacterial Proteins/*antagonists & inhibitors/chemistry/genetics/metabolism
*CRISPR-Cas Systems
Cell Line
Computational Biology/methods
DNA Cleavage
Endonucleases/*antagonists & inhibitors
*Gene Editing
Genome, Bacterial
Humans
Moraxella/*genetics

@article {pmid30119690,
year = {2018},
author = {Han, R and Li, L and Ugalde, AP and Tal, A and Manber, Z and Barbera, EP and Chiara, VD and Elkon, R and Agami, R},
title = {Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescence.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {118},
pmid = {30119690},
issn = {1474-760X},
mesh = {CRISPR-Cas Systems/*genetics ; Cellular Senescence/*genetics ; *Enhancer Elements, Genetic ; Forkhead Transcription Factors/*genetics ; Gene Expression Profiling ; *Genetic Testing ; HEK293 Cells ; Humans ; Models, Biological ; *Oncogenes ; Transcription Factor AP-1/*metabolism ; },
abstract = {BACKGROUND: Functional characterization of non-coding elements in the human genome is a major genomic challenge and the maturation of genome-editing technologies is revolutionizing our ability to achieve this task. Oncogene-induced senescence, a cellular state of irreversible proliferation arrest that is enforced following excessive oncogenic activity, is a major barrier against cancer transformation; therefore, bypassing oncogene-induced senescence is a critical step in tumorigenesis. Here, we aim at further identification of enhancer elements that are required for the establishment of this state.

RESULTS: We first apply genome-wide profiling of enhancer-RNAs (eRNAs) to systematically identify enhancers that are activated upon oncogenic stress. DNA motif analysis of these enhancers indicates AP-1 as a major regulator of the transcriptional program induced by oncogene-induced senescence. We thus constructed a CRISPR-Cas9 sgRNA library designed to target senescence-induced enhancers that are putatively regulated by AP-1 and used it in a functional screen. We identify a critical enhancer that we name EnhAP1-OIS1 and validate that mutating the AP-1 binding site within this element results in oncogene-induced senescence bypass. Furthermore, we identify FOXF1 as the gene regulated by this enhancer and demonstrate that FOXF1 mediates EnhAP1-OIS1 effect on the senescence phenotype.

CONCLUSIONS: Our study elucidates a novel cascade mediated by AP-1 and FOXF1 that regulates oncogene-induced senescence and further demonstrates the power of CRISPR-based functional genomic screens in deciphering the function of non-coding regulatory elements in the genome.},
}

CRISPR-Cas Systems/*genetics
Cellular Senescence/*genetics
*Enhancer Elements, Genetic
Forkhead Transcription Factors/*genetics
Gene Expression Profiling
*Genetic Testing
HEK293 Cells
Humans
Models, Biological
*Oncogenes
Transcription Factor AP-1/*metabolism

@article {pmid30097071,
year = {2018},
author = {Su, J and Huang, YH and Cui, X and Wang, X and Zhang, X and Lei, Y and Xu, J and Lin, X and Chen, K and Lv, J and Goodell, MA and Li, W},
title = {Homeobox oncogene activation by pan-cancer DNA hypermethylation.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {108},
pmid = {30097071},
issn = {1474-760X},
support = {R01 CA193466/CA/NCI NIH HHS/United States ; F99 CA222736/CA/NCI NIH HHS/United States ; R01HG007538/NH/NIH HHS/United States ; R56 DK092883/DK/NIDDK NIH HHS/United States ; U54CA217297/NH/NIH HHS/United States ; CA228140/CA/NCI NIH HHS/United States ; DK092883/NH/NIH HHS/United States ; R01CA193466/NH/NIH HHS/United States ; P50 CA126752/CA/NCI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; CA183252/NH/NIH HHS/United States ; R01 CA183252/CA/NCI NIH HHS/United States ; R01 HG007538/HG/NHGRI NIH HHS/United States ; R01 DK092883/DK/NIDDK NIH HHS/United States ; U54 CA217297/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA (Cytosine-5-)-Methyltransferases/metabolism ; DNA Methylation/*genetics ; *Gene Expression Regulation, Neoplastic ; *Genes, Homeobox ; Humans ; Neoplasms/*genetics ; *Oncogenes ; Promoter Regions, Genetic ; Reference Standards ; },
abstract = {BACKGROUND: Cancers have long been recognized to be not only genetically but also epigenetically distinct from their tissues of origin. Although genetic alterations underlying oncogene upregulation have been well studied, to what extent epigenetic mechanisms, such as DNA methylation, can also induce oncogene expression remains unknown.

RESULTS: Here, through pan-cancer analysis of 4174 genome-wide profiles, including whole-genome bisulfite sequencing data from 30 normal tissues and 35 solid tumors, we discover a strong correlation between gene-body hypermethylation of DNA methylation canyons, defined as broad under-methylated regions, and overexpression of approximately 43% of homeobox genes, many of which are also oncogenes. To gain insights into the cause-and-effect relationship, we use a newly developed dCas9-SunTag-DNMT3A system to methylate genomic sites of interest. The locus-specific hypermethylation of gene-body canyon, but not promoter, of homeobox oncogene DLX1, can directly increase its gene expression.

CONCLUSIONS: Our pan-cancer analysis followed by functional validation reveals DNA hypermethylation as a novel epigenetic mechanism for homeobox oncogene upregulation.},
}

CRISPR-Cas Systems/genetics
DNA (Cytosine-5-)-Methyltransferases/metabolism
DNA Methylation/*genetics
*Gene Expression Regulation, Neoplastic
*Genes, Homeobox
Humans
Neoplasms/*genetics
*Oncogenes
Promoter Regions, Genetic
Reference Standards

@article {pmid30086262,
year = {2018},
author = {Chugh, S and Barkeer, S and Rachagani, S and Nimmakayala, RK and Perumal, N and Pothuraju, R and Atri, P and Mahapatra, S and Thapa, I and Talmon, GA and Smith, LM and Yu, X and Neelamegham, S and Fu, J and Xia, L and Ponnusamy, MP and Batra, SK},
title = {Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice.},
journal = {Gastroenterology},
volume = {155},
number = {5},
pages = {1608-1624},
pmid = {30086262},
issn = {1528-0012},
support = {U01 CA200466/CA/NCI NIH HHS/United States ; R01 HL103411/HL/NHLBI NIH HHS/United States ; R01 CA195586/CA/NCI NIH HHS/United States ; P50 CA127297/CA/NCI NIH HHS/United States ; R01 DK085691/DK/NIDDK NIH HHS/United States ; P30 GM114731/GM/NIGMS NIH HHS/United States ; U01 CA210240/CA/NCI NIH HHS/United States ; R01 CA183459/CA/NCI NIH HHS/United States ; R01 CA210637/CA/NCI NIH HHS/United States ; P01 CA217798/CA/NCI NIH HHS/United States ; },
mesh = {Adenocarcinoma/*etiology/secondary ; Animals ; CRISPR-Cas Systems ; Carcinoma, Pancreatic Ductal ; Cell Proliferation ; Galactosyltransferases/genetics/*physiology ; Glycosylation ; Humans ; Mice ; Mice, Inbred C57BL ; Pancreatic Neoplasms/*etiology/pathology ; },
abstract = {BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1.

METHODS: We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry.

RESULTS: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis.

CONCLUSIONS: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.},
}

Adenocarcinoma/*etiology/secondary
Animals
CRISPR-Cas Systems
Carcinoma, Pancreatic Ductal
Cell Proliferation
Galactosyltransferases/genetics/*physiology
Glycosylation
Humans
Mice
Mice, Inbred C57BL
Pancreatic Neoplasms/*etiology/pathology

@article {pmid30075414,
year = {2018},
author = {Su, X and Wang, S and Su, G and Zheng, Z and Zhang, J and Ma, Y and Liu, Z and Zhou, H and Zhang, Y and Zhang, L},
title = {Production of microhomologous-mediated site-specific integrated LacS gene cow using TALENs.},
journal = {Theriogenology},
volume = {119},
number = {},
pages = {282-288},
doi = {10.1016/j.theriogenology.2018.07.011},
pmid = {30075414},
issn = {1879-3231},
mesh = {Animals ; Animals, Genetically Modified ; Animals, Newborn ; CRISPR-Cas Systems ; Cattle/*genetics ; DNA ; Female ; Gene Editing/methods/*veterinary ; Genetic Engineering/methods ; Genotype ; Transcription Activator-Like Effector Nucleases ; },
abstract = {Gene editing tools (Zinc-Finger Nucleases, ZFN; Transcription Activator-Like Effector Nucleases, TALEN; and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9, CRISPR-Cas9) provide us with a powerful means of performing genetic engineering procedures. A combinational approach that utilizes both somatic cell nuclear transfer (SCNT) and somatic cell gene editing facilitates the generation of genetically engineered animals. However, the associated research has utilized markers and/or selected genes, which constitute a potential threat to biosafety. Microhomologous-mediated end-joining (MMEJ) has showed the utilization of micro-homologous arms (5-25 bp) can mediate exogenous gene insertion. Dairy milk is a major source of nutrition worldwide. However, most people are not capable of optimally utilizing the nutrition in milk because of lactose intolerance. Sulfolobus solfataricus β-glycosidase (LacS) is a lactase derived from the extreme thermophilic archaeon Sulfolobus solfataricus. Our finally aim was to site-specific integrated LacS gene into cow's genome through TALEN-mediated MMEJ and produce low-lactose cow. Firstly, we constructed TALENs vectors which target to the cow's β-casein locus and LacS gene expression vector which contain TALEN reorganization sequence and micro-homologous arms. Then we co-transfected these vectors into fetal derived skin fibroblasts and cultured as monoclone. Positive cell clones were screened using 3' junction PCR amplification and sequencing analysis. The positive cells were used as donors for SCNT and embryo transfer (ET). Lastly, we detected the genotype through PCR of blood genomic DNA. This resulted in a LacS knock-in rate of 0.8% in TALEN-treated cattle fetal fibroblasts. The blastocyst rate of SCNT embryo was 27%. The 3 months pregnancy rate was 20%. Finally, we obtained 1 newborn cow (5%) and verified its genotype. We obtained 1 site-specific marker-free LacS transgenic cow. It provides a basis to solve lactose intolerance by gene engineering breeding. This study also provides us with a new strategy to facilitate gene knock-ins in livestock using techniques that exhibit improved biosafety and intuitive methodologies.},
}

Animals
Animals, Genetically Modified
Animals, Newborn
CRISPR-Cas Systems
Cattle/*genetics
DNA
Female
Gene Editing/methods/*veterinary
Genetic Engineering/methods
Genotype
Transcription Activator-Like Effector Nucleases

@article {pmid30046996,
year = {2018},
author = {Nozawa, K and Hayashi, A and Motohashi, J and Takeo, YH and Matsuda, K and Yuzaki, M},
title = {Cellular and Subcellular Localization of Endogenous Neuroligin-1 in the Cerebellum.},
journal = {Cerebellum (London, England)},
volume = {17},
number = {6},
pages = {709-721},
pmid = {30046996},
issn = {1473-4230},
support = {JP16H06461//Japan Society for the Promotion of Science/ ; JPMJCR13L6//Japan Science and Technology Agency/ ; JP15H05772//Ministry of Education, Culture, Sports, Science and Technology (JP)/ ; },
mesh = {Animals ; Astrocytes/cytology/metabolism ; CRISPR-Cas Systems ; Cell Adhesion Molecules, Neuronal/genetics/*metabolism ; Cerebellum/*cytology/*metabolism ; Epitopes ; Gene Knockdown Techniques ; Genetic Engineering ; HEK293 Cells ; Hemagglutinins/genetics/immunology ; Hippocampus/cytology/metabolism ; Humans ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mice, Inbred ICR ; Mice, Transgenic ; Neurons/cytology/metabolism ; Synapses/metabolism ; },
abstract = {Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in the Nlgn1 gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. Using HA-Nlgn1 mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.},
}

Animals
Astrocytes/cytology/metabolism
CRISPR-Cas Systems
Cell Adhesion Molecules, Neuronal/genetics/*metabolism
Cerebellum/*cytology/*metabolism
Epitopes
Gene Knockdown Techniques
Genetic Engineering
HEK293 Cells
Hemagglutinins/genetics/immunology
Hippocampus/cytology/metabolism
Humans
Mice, Inbred C57BL
Mice, Inbred DBA
Mice, Inbred ICR
Mice, Transgenic
Neurons/cytology/metabolism
Synapses/metabolism

@article {pmid29946130,
year = {2018},
author = {Wang, T and Guan, C and Guo, J and Liu, B and Wu, Y and Xie, Z and Zhang, C and Xing, XH},
title = {Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2475},
pmid = {29946130},
issn = {2041-1723},
support = {NSFC21627812//National Natural Science Foundation of China (National Science Foundation of China)/International ; },
mesh = {Bacteria/drug effects/*genetics/metabolism ; Butanols/toxicity ; *CRISPR-Cas Systems ; Chromosome Mapping ; Escherichia coli/drug effects/genetics/metabolism ; Furaldehyde/toxicity ; Gene Library ; Gene Regulatory Networks ; Genes, Essential ; Genome, Bacterial ; Genomics ; High-Throughput Nucleotide Sequencing ; INDEL Mutation ; Metabolic Networks and Pathways/genetics ; RNA, Bacterial/genetics ; RNA, Guide/genetics ; RNA, Untranslated/genetics ; Transcriptome ; },
abstract = {To fully exploit the microbial genome resources, a high-throughput experimental platform is needed to associate genes with phenotypes at the genome level. We present here a novel method that enables investigation of the cellular consequences of repressing individual transcripts based on the CRISPR interference (CRISPRi) pooled screening in bacteria. We identify rules for guide RNA library design to handle the unique structure of prokaryotic genomes by tiling screening and construct an E. coli genome-scale guide RNA library (~60,000 members) accordingly. We show that CRISPRi outperforms transposon sequencing, the benchmark method in the microbial functional genomics field, when similar library sizes are used or gene length is short. This tool is also effective for mapping phenotypes to non-coding RNAs (ncRNAs), as elucidated by a comprehensive tRNA-fitness map constructed here. Our results establish CRISPRi pooled screening as a powerful tool for mapping complex prokaryotic genetic networks in a precise and high-throughput manner.},
}

Bacteria/drug effects/*genetics/metabolism
Butanols/toxicity
*CRISPR-Cas Systems
Chromosome Mapping
Escherichia coli/drug effects/genetics/metabolism
Furaldehyde/toxicity
Gene Library
Gene Regulatory Networks
Genes, Essential
Genome, Bacterial
Genomics
High-Throughput Nucleotide Sequencing
INDEL Mutation
Metabolic Networks and Pathways/genetics
RNA, Bacterial/genetics
RNA, Guide/genetics
RNA, Untranslated/genetics
Transcriptome

@article {pmid29914939,
year = {2018},
author = {Jokl, EJ and Hughes, GL and Cracknell, T and Pownall, ME and Blanco, G},
title = {Transcriptional upregulation of Bag3, a chaperone-assisted selective autophagy factor, in animal models of KY-deficient hereditary myopathy.},
journal = {Disease models & mechanisms},
volume = {11},
number = {7},
pages = {},
pmid = {29914939},
issn = {1754-8411},
support = {//Wellcome Trust/United Kingdom ; BB/J014443/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; 204829//Wellcome Trust/United Kingdom ; },
mesh = {Adaptor Proteins, Signal Transducing/*genetics/metabolism ; Animals ; Apoptosis Regulatory Proteins/*genetics/metabolism ; *Autophagy ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Disease Models, Animal ; Filamins/metabolism ; Gene Editing ; Mechanotransduction, Cellular ; Mice, Knockout ; Muscle Fibers, Skeletal/metabolism/pathology ; Muscle Proteins/*deficiency/metabolism ; Muscular Diseases/*genetics/*pathology ; Mutagenesis/genetics ; Transcription, Genetic ; Up-Regulation/*genetics ; Zebrafish ; Zebrafish Proteins/*deficiency/genetics/metabolism ; },
abstract = {The importance of kyphoscoliosis peptidase (KY) in skeletal muscle physiology has recently been emphasised by the identification of novel human myopathies associated with KY deficiency. Neither the pathogenic mechanism of KY deficiency nor a specific role for KY in muscle function have been established. However, aberrant localisation of filamin C (FLNC) in muscle fibres has been shown in humans and mice with loss-of-function mutations in the KY gene. FLNC turnover has been proposed to be controlled by chaperone-assisted selective autophagy (CASA), a client-specific and tension-induced pathway that is required for muscle maintenance. Here, we have generated new C2C12 myoblast and zebrafish models of KY deficiency by CRISPR/Cas9 mutagenesis. To obtain insights into the pathogenic mechanism caused by KY deficiency, expression of the co-chaperone BAG3 and other CASA factors was analyzed in the cellular, zebrafish and ky/ky mouse models. Ky-deficient C2C12-derived clones show trends of higher transcription of CASA factors in differentiated myotubes. The ky-deficient zebrafish model (kyyo1/kyyo1) lacks overt signs of pathology, but shows significantly increased bag3 and flnca/b expression in embryos and adult muscle. Additionally, kyyo1/kyyo1 embryos challenged by swimming in viscous media show an inability to further increase expression of these factors in contrast with wild-type controls. The ky/ky mouse shows elevated expression of Bag3 in the non-pathological exterior digitorum longus (EDL) and evidence of impaired BAG3 turnover in the pathological soleus. Thus, upregulation of CASA factors appears to be an early and primary molecular hallmark of KY deficiency.},
}

Adaptor Proteins, Signal Transducing/*genetics/metabolism
Animals
Apoptosis Regulatory Proteins/*genetics/metabolism
*Autophagy
Base Sequence
CRISPR-Cas Systems/genetics
Cell Differentiation
Cell Line
Disease Models, Animal
Filamins/metabolism
Gene Editing
Mechanotransduction, Cellular
Mice, Knockout
Muscle Fibers, Skeletal/metabolism/pathology
Muscle Proteins/*deficiency/metabolism
Muscular Diseases/*genetics/*pathology
Mutagenesis/genetics
Transcription, Genetic
Up-Regulation/*genetics
Zebrafish
Zebrafish Proteins/*deficiency/genetics/metabolism

@article {pmid29871865,
year = {2018},
author = {Sui, T and Lau, YS and Liu, D and Liu, T and Xu, L and Gao, Y and Lai, L and Li, Z and Han, R},
title = {A novel rabbit model of Duchenne muscular dystrophy generated by CRISPR/Cas9.},
journal = {Disease models & mechanisms},
volume = {11},
number = {6},
pages = {},
pmid = {29871865},
issn = {1754-8411},
support = {R01 HL116546/HL/NHLBI NIH HHS/United States ; R01 AR064241/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cardiomyopathies/genetics/pathology/physiopathology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Creatine Kinase, MM Form/blood ; Disease Models, Animal ; Disease Progression ; Dystrophin/*genetics/metabolism ; Fibrosis ; Gene Editing/*methods ; Genetic Predisposition to Disease ; Muscle, Skeletal/metabolism/pathology/physiopathology ; Muscular Dystrophy, Duchenne/*genetics/metabolism/pathology/physiopathology ; Myocardial Contraction ; Necrosis ; Phenotype ; Rabbits ; Stroke Volume ; Time Factors ; },
abstract = {Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disorder caused by mutations in the dystrophin gene, with an incidence of 1 in 3500 in new male births. Mdx mice are widely used as an animal model for DMD. However, these mice do not faithfully recapitulate DMD patients in many aspects, rendering the preclinical findings in this model questionable. Although larger animal models of DMD, such as dogs and pigs, have been generated, usage of these animals is expensive and only limited to several facilities in the world. Here, we report the generation of a rabbit model of DMD by co-injection of Cas9 mRNA and sgRNA targeting exon 51 into rabbit zygotes. The DMD knockout (KO) rabbits exhibit the typical phenotypes of DMD, including severely impaired physical activity, elevated serum creatine kinase levels, and progressive muscle necrosis and fibrosis. Moreover, clear pathology was also observed in the diaphragm and heart at 5 months of age, similar to DMD patients. Echocardiography recording showed that the DMD KO rabbits had chamber dilation with decreased ejection fraction and fraction shortening. In conclusion, this novel rabbit DMD model generated with the CRISPR/Cas9 system mimics the histopathological and functional defects in DMD patients, and could be valuable for preclinical studies.This article has an associated First Person interview with the first author of the paper.},
}

Animals
Animals, Genetically Modified
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
Cardiomyopathies/genetics/pathology/physiopathology
*Clustered Regularly Interspaced Short Palindromic Repeats
Creatine Kinase, MM Form/blood
Disease Models, Animal
Disease Progression
Dystrophin/*genetics/metabolism
Fibrosis
Gene Editing/*methods
Genetic Predisposition to Disease
Muscle, Skeletal/metabolism/pathology/physiopathology
Muscular Dystrophy, Duchenne/*genetics/metabolism/pathology/physiopathology
Myocardial Contraction
Necrosis
Phenotype
Rabbits
Stroke Volume
Time Factors

@article {pmid29867139,
year = {2018},
author = {Riesenberg, S and Maricic, T},
title = {Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2164},
pmid = {29867139},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; DNA Breaks, Double-Stranded ; DNA Repair/*drug effects/genetics ; *Gene Editing ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/drug effects/metabolism ; K562 Cells ; Models, Genetic ; Mutation ; Pluripotent Stem Cells/*drug effects/metabolism ; Small Molecule Libraries/*pharmacology ; },
abstract = {A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks which are then repaired by homology-directed repair using DNA donor molecules carrying desired mutations. Using a mixture of small molecules, the "CRISPY" mix, we achieve a 2.8- to 7.2-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes or of gene encoding a blue fluorescent protein in 27% of cells, to our knowledge the highest editing efficiency in human induced pluripotent stem cells described to date. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.3- to 4.0-fold, allowing almost 20% of chromosomes to be edited. The components of the CRISPY mix do not always increase the editing efficiency in the immortalized or primary cell lines tested, suggesting that employed repair pathways are cell-type specific.},
}

Animals
CRISPR-Cas Systems
Cell Line
DNA Breaks, Double-Stranded
DNA Repair/*drug effects/genetics
*Gene Editing
HEK293 Cells
Humans
Induced Pluripotent Stem Cells/drug effects/metabolism
K562 Cells
Models, Genetic
Mutation
Pluripotent Stem Cells/*drug effects/metabolism
Small Molecule Libraries/*pharmacology

@article {pmid29856759,
year = {2018},
author = {Thomenius, MJ and Totman, J and Harvey, D and Mitchell, LH and Riera, TV and Cosmopoulos, K and Grassian, AR and Klaus, C and Foley, M and Admirand, EA and Jahic, H and Majer, C and Wigle, T and Jacques, SL and Gureasko, J and Brach, D and Lingaraj, T and West, K and Smith, S and Rioux, N and Waters, NJ and Tang, C and Raimondi, A and Munchhof, M and Mills, JE and Ribich, S and Porter Scott, M and Kuntz, KW and Janzen, WP and Moyer, M and Smith, JJ and Chesworth, R and Copeland, RA and Boriack-Sjodin, PA},
title = {Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.},
journal = {PloS one},
volume = {13},
number = {6},
pages = {e0197372},
pmid = {29856759},
issn = {1932-6203},
mesh = {A549 Cells ; Adenocarcinoma of Lung/*drug therapy/genetics/pathology ; CRISPR-Cas Systems ; Carcinogenesis/drug effects/*genetics ; Cell Proliferation/drug effects ; Enzyme Inhibitors/pharmacology ; Histone-Lysine N-Methyltransferase/antagonists & inhibitors/chemistry/*genetics ; Humans ; Methylation/drug effects ; Methyltransferases/antagonists & inhibitors ; RNA Interference ; Small Molecule Libraries/pharmacology ; },
abstract = {A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.},
}

A549 Cells
Adenocarcinoma of Lung/*drug therapy/genetics/pathology
CRISPR-Cas Systems
Carcinogenesis/drug effects/*genetics
Cell Proliferation/drug effects
Enzyme Inhibitors/pharmacology
Histone-Lysine N-Methyltransferase/antagonists & inhibitors/chemistry/*genetics
Humans
Methylation/drug effects
Methyltransferases/antagonists & inhibitors
RNA Interference
Small Molecule Libraries/pharmacology

@article {pmid29804684,
year = {2018},
author = {McKinley, KL},
title = {Employing CRISPR/Cas9 genome engineering to dissect the molecular requirements for mitosis.},
journal = {Methods in cell biology},
volume = {144},
number = {},
pages = {75-105},
doi = {10.1016/bs.mcb.2018.03.003},
pmid = {29804684},
issn = {0091-679X},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Editing/*methods ; Genetic Loci ; Genome/*genetics ; Humans ; Mitosis/*genetics ; RNA, Guide/metabolism ; },
abstract = {The faithful execution of cell division requires the coordinated action of hundreds of gene products. Precisely perturbing these gene products in cells is central to understanding their functions during normal cell division, and the contributions of their disruption to disease. Here, we describe experimental approaches for using CRISPR/Cas9 for gene disruption and modification, with a focus on human cell culture. We describe strategies for inducible gene disruption to generate acute knockouts of essential cell division genes, which can be modified for the chronic elimination of nonessential genes. We also describe strategies for modifying the genome to generate protein fusions to report on and modify protein behavior. These tools facilitate investigation of protein function, dissection of protein assembly networks, and analyses of disease-associated mutations.},
}

Animals
Base Sequence
CRISPR-Associated Protein 9/*genetics/*metabolism
CRISPR-Cas Systems/*genetics
Cell Line
Gene Editing/*methods
Genetic Loci
Genome/*genetics
Humans
Mitosis/*genetics
RNA, Guide/metabolism

@article {pmid29804670,
year = {2018},
author = {Hattersley, N and Lara-Gonzalez, P and Cheerambathur, D and Gomez-Cavazos, JS and Kim, T and Prevo, B and Khaliullin, R and Lee, KY and Ohta, M and Green, R and Oegema, K and Desai, A},
title = {Employing the one-cell C. elegans embryo to study cell division processes.},
journal = {Methods in cell biology},
volume = {144},
number = {},
pages = {185-231},
doi = {10.1016/bs.mcb.2018.03.008},
pmid = {29804670},
issn = {0091-679X},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Caenorhabditis elegans/*embryology/genetics ; *Cell Division ; Cytological Techniques/*methods ; Embryo, Nonmammalian/*cytology ; Genetic Engineering ; Microtubules/metabolism ; Mutation/genetics ; RNA Interference ; Reproducibility of Results ; Transgenes ; },
abstract = {The one-cell Caenorhabditis elegans embryo offers many advantages for mechanistic analysis of cell division processes. Conservation of key genes and pathways involved in cell division makes findings in C. elegans broadly relevant. A key technical advantage of this system is the ability to penetrantly deplete essential gene products by RNA interference (RNAi) and replace them with wild-type or mutant versions expressed at endogenous levels from single copy RNAi-resistant transgene insertions. This ability to precisely perturb essential genes is complemented by the inherently highly reproducible nature of the zygotic division that facilitates development of quantitative imaging assays. Here, we detail approaches to generate targeted single copy transgene insertions that are RNAi-resistant, to engineer variants of individual genes employing transgene insertions as well as at the endogenous locus, and to in situ tag genes with fluorophores/purification tags. We also describe imaging assays and common image analysis tools employed to quantitatively monitor phenotypic effects of specific perturbations on meiotic and mitotic chromosome segregation, centrosome assembly/function, and cortical dynamics/cytokinesis.},
}

Alleles
Animals
CRISPR-Cas Systems/genetics
Caenorhabditis elegans/*embryology/genetics
*Cell Division
Cytological Techniques/*methods
Embryo, Nonmammalian/*cytology
Genetic Engineering
Microtubules/metabolism
Mutation/genetics
RNA Interference
Reproducibility of Results
Transgenes

@article {pmid29804665,
year = {2018},
author = {Lambrus, BG and Moyer, TC and Holland, AJ},
title = {Applying the auxin-inducible degradation system for rapid protein depletion in mammalian cells.},
journal = {Methods in cell biology},
volume = {144},
number = {},
pages = {107-135},
doi = {10.1016/bs.mcb.2018.03.004},
pmid = {29804665},
issn = {0091-679X},
support = {R01 GM114119/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Line ; DNA Cleavage ; Humans ; Indoleacetic Acids/*pharmacology ; Mammals/*metabolism ; Proteolysis/*drug effects ; RNA, Guide/metabolism ; Reproducibility of Results ; Transduction, Genetic ; },
abstract = {The ability to deplete a protein of interest is critical for dissecting cellular processes. Traditional methods of protein depletion are often slow acting, which can be problematic when characterizing a cellular process that occurs within a short period of time, such as mitosis. Furthermore, these methods are usually not reversible. Recent advances to achieve protein depletion function by inducibly trafficking proteins of interest to an endogenous E3 ubiquitin ligase complex to promote ubiquitination and subsequent degradation by the proteasome. One of these systems, the auxin-inducible degron (AID) system, has been shown to permit rapid and inducible degradation of AID-tagged target proteins in mammalian cells. The AID system can control the abundance of a diverse set of cellular proteins, including those contained within protein complexes, and is active in all phases of the cell cycle. Here we discuss considerations for the successful implementation of the AID system and describe a protocol using CRISPR/Cas9 to achieve biallelic insertion of an AID in human cells. This method can also be adapted to insert other tags, such as fluorescent proteins, at defined genomic locations.},
}

Animals
Base Sequence
CRISPR-Cas Systems/genetics
Cell Line
DNA Cleavage
Humans
Indoleacetic Acids/*pharmacology
Mammals/*metabolism
Proteolysis/*drug effects
RNA, Guide/metabolism
Reproducibility of Results
Transduction, Genetic

@article {pmid29802289,
year = {2018},
author = {Incontro, S and Díaz-Alonso, J and Iafrati, J and Vieira, M and Asensio, CS and Sohal, VS and Roche, KW and Bender, KJ and Nicoll, RA},
title = {The CaMKII/NMDA receptor complex controls hippocampal synaptic transmission by kinase-dependent and independent mechanisms.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2069},
pmid = {29802289},
issn = {2041-1723},
support = {R01 GM124035/GM/NIGMS NIH HHS/United States ; R15 GM116096/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics/*metabolism ; Cell Membrane/metabolism ; Female ; HEK293 Cells ; Hippocampus/cytology/*physiology ; Humans ; Long-Term Potentiation/physiology ; Mice ; Mice, Knockout ; Neurons/cytology/physiology ; Phosphorylation ; Rats ; Receptors, Glutamate/metabolism ; Receptors, N-Methyl-D-Aspartate/*metabolism ; Signal Transduction/physiology ; Synapses/physiology ; Synaptic Transmission/*physiology ; },
abstract = {CaMKII is one of the most studied synaptic proteins, but many critical issues regarding its role in synaptic function remain unresolved. Using a CRISPR-based system to delete CaMKII and replace it with mutated forms in single neurons, we have rigorously addressed its various synaptic roles. In brief, basal AMPAR and NMDAR synaptic transmission both require CaMKIIα, but not CaMKIIβ, indicating that, even in the adult, synaptic transmission is determined by the ongoing action of CaMKIIα. While AMPAR transmission requires kinase activity, NMDAR transmission does not, implying a scaffolding role for the CaMKII protein instead. LTP is abolished in the absence of CaMKIIα and/or CaMKIIβ and with an autophosphorylation impaired CaMKIIα (T286A). With the exception of NMDAR synaptic currents, all aspects of CaMKIIα signaling examined require binding to the NMDAR, emphasizing the essential role of this receptor as a master synaptic signaling hub.},
}

Animals
CRISPR-Cas Systems
Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics/*metabolism
Cell Membrane/metabolism
Female
HEK293 Cells
Hippocampus/cytology/*physiology
Humans
Long-Term Potentiation/physiology
Mice
Mice, Knockout
Neurons/cytology/physiology
Phosphorylation
Rats
Receptors, Glutamate/metabolism
Receptors, N-Methyl-D-Aspartate/*metabolism
Signal Transduction/physiology
Synapses/physiology
Synaptic Transmission/*physiology

@article {pmid29799876,
year = {2018},
author = {Hegde, M and Strand, C and Hanna, RE and Doench, JG},
title = {Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens.},
journal = {PloS one},
volume = {13},
number = {5},
pages = {e0197547},
pmid = {29799876},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Barcoding, Taxonomic/*methods ; Genetic Vectors ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Lentivirus/genetics ; Polymerase Chain Reaction/*methods ; RNA, Guide/*genetics ; Recombination, Genetic ; },
abstract = {Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.},
}

*CRISPR-Cas Systems
Cell Line, Tumor
DNA Barcoding, Taxonomic/*methods
Genetic Vectors
High-Throughput Nucleotide Sequencing/*methods
Humans
Lentivirus/genetics
Polymerase Chain Reaction/*methods
RNA, Guide/*genetics
Recombination, Genetic

@article {pmid29795191,
year = {2018},
author = {Zhao, H and Wang, X and Jia, Y and Minkenberg, B and Wheatley, M and Fan, J and Jia, MH and Famoso, A and Edwards, JD and Wamishe, Y and Valent, B and Wang, GL and Yang, Y},
title = {The rice blast resistance gene Ptr encodes an atypical protein required for broad-spectrum disease resistance.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2039},
pmid = {29795191},
issn = {2041-1723},
mesh = {Armadillo Domain Proteins/*genetics/immunology ; CRISPR-Cas Systems ; Chromosome Mapping ; Disease Resistance/*genetics ; Genes, Plant/*immunology ; Host Microbial Interactions/genetics/immunology ; Magnaporthe/immunology/pathogenicity ; Mutagenesis ; Oryza/microbiology/*physiology ; Plant Diseases/*genetics/microbiology ; Plant Immunity/genetics ; Plant Proteins/*genetics/immunology ; Plants, Genetically Modified ; Protein Isoforms/genetics/immunology ; Receptors, Cytoplasmic and Nuclear/*genetics/immunology ; Sequence Analysis, DNA ; },
abstract = {Plant resistance genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. Here we show that Ptr is an atypical resistance gene encoding a protein with four Armadillo repeats. Ptr is required for broad-spectrum blast resistance mediated by the NLR R gene Pi-ta and by the associated R gene Pi-ta2. Ptr is expressed constitutively and encodes two isoforms that are mainly localized in the cytoplasm. A two base pair deletion within the Ptr coding region in the fast neutron-generated mutant line M2354 creates a truncated protein, resulting in susceptibility to M. oryzae. Targeted mutation of Ptr in a resistant cultivar using CRISPR/Cas9 leads to blast susceptibility, further confirming its resistance function. The cloning of Ptr may aid in the development of broad spectrum blast resistant rice.},
}

Armadillo Domain Proteins/*genetics/immunology
CRISPR-Cas Systems
Chromosome Mapping
Disease Resistance/*genetics
Genes, Plant/*immunology
Host Microbial Interactions/genetics/immunology
Magnaporthe/immunology/pathogenicity
Mutagenesis
Oryza/microbiology/*physiology
Plant Diseases/*genetics/microbiology
Plant Immunity/genetics
Plant Proteins/*genetics/immunology
Plants, Genetically Modified
Protein Isoforms/genetics/immunology
Receptors, Cytoplasmic and Nuclear/*genetics/immunology
Sequence Analysis, DNA

@article {pmid29789620,
year = {2018},
author = {Yang, TT and Chong, WM and Wang, WJ and Mazo, G and Tanos, B and Chen, Z and Tran, TMN and Chen, YD and Weng, RR and Huang, CE and Jane, WN and Tsou, MB and Liao, JC},
title = {Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2023},
pmid = {29789620},
issn = {2041-1723},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; P30 DK074038/DK/NIDDK NIH HHS/United States ; R01 GM088253/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/*chemistry/genetics/metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism/*ultrastructure ; Cell Line ; Centrioles/metabolism/*ultrastructure ; Cilia/metabolism/*ultrastructure ; Epithelial Cells/metabolism/ultrastructure ; Gene Editing ; Gene Expression ; HEK293 Cells ; Humans ; Microtubule Proteins/genetics/metabolism/*ultrastructure ; Microtubule-Associated Proteins/genetics/metabolism/*ultrastructure ; Molecular Imaging ; Protein Multimerization ; Retinal Pigment Epithelium/metabolism/ultrastructure ; Sodium Channels/genetics/metabolism/*ultrastructure ; },
abstract = {Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.},
}

Adaptor Proteins, Signal Transducing/*chemistry/genetics/metabolism
CRISPR-Cas Systems
Cell Cycle Proteins/genetics/metabolism/*ultrastructure
Cell Line
Centrioles/metabolism/*ultrastructure
Cilia/metabolism/*ultrastructure
Epithelial Cells/metabolism/ultrastructure
Gene Editing
Gene Expression
HEK293 Cells
Humans
Microtubule Proteins/genetics/metabolism/*ultrastructure
Microtubule-Associated Proteins/genetics/metabolism/*ultrastructure
Molecular Imaging
Protein Multimerization
Retinal Pigment Epithelium/metabolism/ultrastructure
Sodium Channels/genetics/metabolism/*ultrastructure

@article {pmid29773790,
year = {2018},
author = {Miki, D and Zhang, W and Zeng, W and Feng, Z and Zhu, JK},
title = {CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {1967},
pmid = {29773790},
issn = {2041-1723},
mesh = {Amino Acid Substitution/genetics ; Arabidopsis/*genetics ; Arabidopsis Proteins/genetics ; *CRISPR-Cas Systems ; Gene Knock-In Techniques/*methods ; Gene Targeting/*methods ; Genome, Plant/genetics ; Nuclear Proteins/genetics ; Plants, Genetically Modified/genetics ; RNA, Guide/genetics ; Transformation, Genetic/genetics ; },
abstract = {Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.},
}

Amino Acid Substitution/genetics
Arabidopsis/*genetics
Arabidopsis Proteins/genetics
*CRISPR-Cas Systems
Gene Knock-In Techniques/*methods
Gene Targeting/*methods
Genome, Plant/genetics
Nuclear Proteins/genetics
Plants, Genetically Modified/genetics
RNA, Guide/genetics
Transformation, Genetic/genetics

@article {pmid29748565,
year = {2018},
author = {Pettitt, SJ and Krastev, DB and Brandsma, I and Dréan, A and Song, F and Aleksandrov, R and Harrell, MI and Menon, M and Brough, R and Campbell, J and Frankum, J and Ranes, M and Pemberton, HN and Rafiq, R and Fenwick, K and Swain, A and Guettler, S and Lee, JM and Swisher, EM and Stoynov, S and Yusa, K and Ashworth, A and Lord, CJ},
title = {Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {1849},
pmid = {29748565},
issn = {2041-1723},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Aged ; Animals ; BRCA1 Protein/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Mutational Analysis/methods ; Drug Resistance, Neoplasm/*genetics ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mouse Embryonic Stem Cells ; Mutagenesis ; Neoplasms/*drug therapy/genetics/pathology ; Phthalazines/pharmacology/therapeutic use ; Point Mutation ; Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors/*genetics ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology/therapeutic use ; Precision Medicine/methods ; Whole Genome Sequencing/methods ; Xenograft Model Antitumor Assays ; Zinc Fingers/genetics ; },
abstract = {Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.},
}

Aged
Animals
BRCA1 Protein/genetics
CRISPR-Cas Systems
Cell Line, Tumor
DNA Mutational Analysis/methods
Drug Resistance, Neoplasm/*genetics
Female
Humans
Mice
Mice, Inbred BALB C
Mice, Nude
Mouse Embryonic Stem Cells
Mutagenesis
Neoplasms/*drug therapy/genetics/pathology
Phthalazines/pharmacology/therapeutic use
Point Mutation
Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors/*genetics
Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology/therapeutic use
Precision Medicine/methods
Whole Genome Sequencing/methods
Xenograft Model Antitumor Assays
Zinc Fingers/genetics

@article {pmid29717121,
year = {2018},
author = {Zou, X and Owusu, M and Harris, R and Jackson, SP and Loizou, JI and Nik-Zainal, S},
title = {Validating the concept of mutational signatures with isogenic cell models.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {1744},
pmid = {29717121},
issn = {2041-1723},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Algorithms ; CRISPR-Cas Systems ; Cell Line ; DNA Damage ; DNA Repair/genetics ; Humans ; *Models, Biological ; *Mutation ; Neoplasms/*genetics ; },
abstract = {The diversity of somatic mutations in human cancers can be decomposed into individual mutational signatures, patterns of mutagenesis that arise because of DNA damage and DNA repair processes that have occurred in cells as they evolved towards malignancy. Correlations between mutational signatures and environmental exposures, enzymatic activities and genetic defects have been described, but human cancers are not ideal experimental systems-the exposures to different mutational processes in a patient's lifetime are uncontrolled and any relationships observed can only be described as an association. Here, we demonstrate the proof-of-principle that it is possible to recreate cancer mutational signatures in vitro using CRISPR-Cas9-based gene-editing experiments in an isogenic human-cell system. We provide experimental and algorithmic methods to discover mutational signatures generated under highly experimentally-controlled conditions. Our in vitro findings strikingly recapitulate in vivo observations of cancer data, fundamentally validating the concept of (particularly) endogenously-arising mutational signatures.},
}

Algorithms
CRISPR-Cas Systems
Cell Line
DNA Damage
DNA Repair/genetics
Humans
*Models, Biological
*Mutation
Neoplasms/*genetics

@article {pmid29716531,
year = {2018},
author = {Hegge, B and Sjøttem, E and Mikkola, I},
title = {Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress.},
journal = {BMC cancer},
volume = {18},
number = {1},
pages = {496},
pmid = {29716531},
issn = {1471-2407},
mesh = {Antineoplastic Agents/pharmacology ; Biomarkers ; Brain Neoplasms/*genetics/*metabolism ; CRISPR-Cas Systems ; Cell Cycle/*genetics ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Drug Resistance, Neoplasm/genetics ; Gene Expression ; *Gene Knockout Techniques ; Gene Targeting ; Genes, Reporter ; Genes, Tumor Suppressor ; Glioblastoma/*genetics/*metabolism ; Humans ; *Oxidative Stress ; PAX6 Transcription Factor/*genetics ; },
abstract = {BACKGROUND: The transcription factor PAX6 is expressed in various cancers. In anaplastic astrocytic glioma, PAX6 expression is inversely related to tumor grade, resulting in low PAX6 expression in Glioblastoma, the highest-grade astrocytic glioma. The aim of the present study was to develop a PAX6 knock out cell line as a tool for molecular studies of the roles PAX6 have in attenuating glioblastoma tumor progression.

METHODS: The CRISPR-Cas9 technique was used to knock out PAX6 in U251 N cells. Viral transduction of a doxycycline inducible EGFP-PAX6 expression vector was used to re-introduce (rescue) PAX6 expression in the PAX6 knock out cells. The knock out and rescued cells were rigorously characterized by analyzing morphology, proliferation, colony forming abilities and responses to oxidative stress and chemotherapeutic agents.

RESULTS: The knock out cells had increased proliferation and colony forming abilities compared to wild type cells, consistent with clinical observations indicating that PAX6 functions as a tumor-suppressor. Cell cycle distribution and sensitivity to H2O2 induced oxidative stress were further studied, as well as the effect of different chemotherapeutic agents. For the PAX6 knock out cells, the percentage of cells in G2/M phase increased compared to PAX6 control cells, indicating that PAX6 keeps U251 N cells in the G1 phase of the cell cycle. Interestingly, PAX6 knock out cells were more resilient to H2O2 induced oxidative stress than wild type cells. Chemotherapy treatment is known to generate oxidative stress, hence the effect of several chemotherapeutic agents were tested. We discovered interesting differences in the sensitivity to chemotherapeutic drugs (Temozolomide, Withaferin A and Sulforaphane) between the PAX6 expressing and non-expressing cells.

CONCLUSIONS: The U251 N PAX6 knock out cell lines generated can be used as a tool to study the molecular functions and mechanisms of PAX6 as a tumor suppressor with regard to tumor progression and treatment of glioblastoma.},
}

Antineoplastic Agents/pharmacology
Biomarkers
Brain Neoplasms/*genetics/*metabolism
CRISPR-Cas Systems
Cell Cycle/*genetics
Cell Line, Tumor
Cell Movement
Cell Proliferation
Drug Resistance, Neoplasm/genetics
Gene Expression
*Gene Knockout Techniques
Gene Targeting
Genes, Reporter
Genes, Tumor Suppressor
Glioblastoma/*genetics/*metabolism
Humans
*Oxidative Stress
PAX6 Transcription Factor/*genetics

@article {pmid29712893,
year = {2018},
author = {Peeva, V and Blei, D and Trombly, G and Corsi, S and Szukszto, MJ and Rebelo-Guiomar, P and Gammage, PA and Kudin, AP and Becker, C and Altmüller, J and Minczuk, M and Zsurka, G and Kunz, WS},
title = {Linear mitochondrial DNA is rapidly degraded by components of the replication machinery.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {1727},
pmid = {29712893},
issn = {2041-1723},
mesh = {Base Sequence ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; *DNA Cleavage ; DNA Helicases/genetics/metabolism ; DNA Polymerase gamma/genetics/metabolism ; *DNA Replication ; DNA, Mitochondrial/*genetics/metabolism ; Deoxyribonucleases, Type II Site-Specific/genetics/metabolism ; Electron Transport Complex IV/genetics/metabolism ; Exodeoxyribonucleases/genetics/metabolism ; Gene Editing/*methods ; Genetic Therapy ; HEK293 Cells ; Humans ; Mitochondria/*genetics/metabolism/pathology ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5'-3'exonuclease MGME1, elimination of the 3'-5'exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1.},
}

Base Sequence
CRISPR-Cas Systems
DNA Breaks, Double-Stranded
*DNA Cleavage
DNA Helicases/genetics/metabolism
DNA Polymerase gamma/genetics/metabolism
*DNA Replication
DNA, Mitochondrial/*genetics/metabolism
Deoxyribonucleases, Type II Site-Specific/genetics/metabolism
Electron Transport Complex IV/genetics/metabolism
Exodeoxyribonucleases/genetics/metabolism
Gene Editing/*methods
Genetic Therapy
HEK293 Cells
Humans
Mitochondria/*genetics/metabolism/pathology
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid29706346,
year = {2018},
author = {Chen, XF and Zhu, DL and Yang, M and Hu, WX and Duan, YY and Lu, BJ and Rong, Y and Dong, SS and Hao, RH and Chen, JB and Chen, YX and Yao, S and Thynn, HN and Guo, Y and Yang, TL},
title = {An Osteoporosis Risk SNP at 1p36.12 Acts as an Allele-Specific Enhancer to Modulate LINC00339 Expression via Long-Range Loop Formation.},
journal = {American journal of human genetics},
volume = {102},
number = {5},
pages = {776-793},
pmid = {29706346},
issn = {1537-6605},
mesh = {*Alleles ; Asian Continental Ancestry Group/genetics ; Base Sequence ; Bone Density/genetics ; Bone and Bones/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Chromatin/metabolism ; Chromosomes, Human, Pair 1/*genetics ; *Enhancer Elements, Genetic ; *Gene Expression Regulation ; Genome-Wide Association Study ; Humans ; Models, Genetic ; *Nucleic Acid Conformation ; Osteoporosis/*genetics ; Polymorphism, Single Nucleotide/*genetics ; Promoter Regions, Genetic ; Protein Binding ; Quantitative Trait Loci/genetics ; RNA, Long Noncoding/chemistry/*genetics ; Reproducibility of Results ; Risk Factors ; Transcription Factors/metabolism ; },
abstract = {Genome-wide association studies (GWASs) have reproducibly associated variants within intergenic regions of 1p36.12 locus with osteoporosis, but the functional roles underlying these noncoding variants are unknown. Through an integrative functional genomic and epigenomic analyses, we prioritized rs6426749 as a potential causal SNP for osteoporosis at 1p36.12. Dual-luciferase assay and CRISPR/Cas9 experiments demonstrate that rs6426749 acts as a distal allele-specific enhancer regulating expression of a lncRNA (LINC00339) (∼360 kb) via long-range chromatin loop formation and that this loop is mediated by CTCF occupied near rs6426749 and LINC00339 promoter region. Specifically, rs6426749-G allele can bind transcription factor TFAP2A, which efficiently elevates the enhancer activity and increases LINC00339 expression. Downregulation of LINC00339 significantly increases the expression of CDC42 in osteoblast cells, which is a pivotal regulator involved in bone metabolism. Our study provides mechanistic insight into how a noncoding SNP affects osteoporosis by long-range interaction, a finding that could indicate promising therapeutic targets for osteoporosis.},
}

*Alleles
Asian Continental Ancestry Group/genetics
Base Sequence
Bone Density/genetics
Bone and Bones/metabolism
CRISPR-Cas Systems/genetics
Cell Line
Chromatin/metabolism
Chromosomes, Human, Pair 1/*genetics
*Enhancer Elements, Genetic
*Gene Expression Regulation
Genome-Wide Association Study
Humans
Models, Genetic
*Nucleic Acid Conformation
Osteoporosis/*genetics
Polymorphism, Single Nucleotide/*genetics
Promoter Regions, Genetic
Protein Binding
Quantitative Trait Loci/genetics
RNA, Long Noncoding/chemistry/*genetics
Reproducibility of Results
Risk Factors
Transcription Factors/metabolism

@article {pmid29694924,
year = {2018},
author = {Lou, W and Reynolds, CA and Li, Y and Liu, J and Hüttemann, M and Schlame, M and Stevenson, D and Strathdee, D and Greenberg, ML},
title = {Loss of tafazzin results in decreased myoblast differentiation in C2C12 cells: A myoblast model of Barth syndrome and cardiolipin deficiency.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {1863},
number = {8},
pages = {857-865},
pmid = {29694924},
issn = {1388-1981},
support = {R01 HL117880/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Barth Syndrome/genetics/*pathology ; CRISPR-Cas Systems ; Cardiolipins/*metabolism ; Cell Differentiation/*genetics ; Cell Line ; Gene Knockout Techniques ; Humans ; Lysophospholipids/*metabolism ; Mice ; Mitochondria/metabolism/pathology ; Models, Biological ; Muscle, Skeletal/cytology/metabolism/pathology ; Myoblasts/cytology/metabolism/*pathology ; Transcription Factors/genetics/*metabolism ; },
abstract = {Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.},
}

Animals
Barth Syndrome/genetics/*pathology
CRISPR-Cas Systems
Cardiolipins/*metabolism
Cell Differentiation/*genetics
Cell Line
Gene Knockout Techniques
Humans
Lysophospholipids/*metabolism
Mice
Mitochondria/metabolism/pathology
Models, Biological
Muscle, Skeletal/cytology/metabolism/pathology
Myoblasts/cytology/metabolism/*pathology
Transcription Factors/genetics/*metabolism

@article {pmid29679004,
year = {2018},
author = {Li, Z and Zhang, J and Liu, X and Li, S and Wang, Q and Di Chen, and Hu, Z and Yu, T and Ding, J and Li, J and Yao, M and Fan, J and Huang, S and Gao, Q and Zhao, Y and He, X},
title = {The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {1572},
pmid = {29679004},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carcinoma, Hepatocellular/genetics/*pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; Female ; Gene Expression Regulation, Neoplastic/*genetics ; HEK293 Cells ; Humans ; Liver Neoplasms/genetics/*pathology ; Mice ; Mice, Inbred BALB C ; Neoplasm Invasiveness/genetics ; Neoplasm Transplantation ; Oncogenes/genetics ; Prognosis ; Proteasome Endopeptidase Complex/metabolism ; Protein-Arginine N-Methyltransferases/*metabolism ; RNA, Long Noncoding/*genetics ; Transplantation, Heterologous ; },
abstract = {Recurrent chromosomal aberrations have led to the discovery of oncogenes or tumour suppressors involved in carcinogenesis. Here we characterized an oncogenic long intergenic non-coding RNA in the frequent DNA-gain regions in hepatocellular carcinoma (HCC), LINC01138 (long intergenic non-coding RNA located on 1q21.2). The LINC01138 locus is frequently amplified in HCC; the LINC01138 transcript is stabilized by insulin like growth factor-2 mRNA-binding proteins 1/3 (IGF2BP1/IGF2BP3) and is associated with the malignant features and poor outcomes of HCC patients. LINC01138 acts as an oncogenic driver that promotes cell proliferation, tumorigenicity, tumour invasion and metastasis by physically interacting with arginine methyltransferase 5 (PRMT5) and enhancing its protein stability by blocking ubiquitin/proteasome-dependent degradation in HCC. The discovery of LINC01138, a promising prognostic indicator, provides insight into the molecular pathogenesis of HCC, and the LINC01138/PRMT5 axis is an ideal therapeutic target for HCC treatment.},
}

Animals
CRISPR-Cas Systems/genetics
Carcinoma, Hepatocellular/genetics/*pathology
Cell Line, Tumor
Cell Proliferation/genetics
Female
Gene Expression Regulation, Neoplastic/*genetics
HEK293 Cells
Humans
Liver Neoplasms/genetics/*pathology
Mice
Mice, Inbred BALB C
Neoplasm Invasiveness/genetics
Neoplasm Transplantation
Oncogenes/genetics
Prognosis
Proteasome Endopeptidase Complex/metabolism
Protein-Arginine N-Methyltransferases/*metabolism
RNA, Long Noncoding/*genetics
Transplantation, Heterologous

@article {pmid29676246,
year = {2018},
author = {Mohanty, BK and Kushner, SR},
title = {Enzymes Involved in Posttranscriptional RNA Metabolism in Gram-Negative Bacteria.},
journal = {Microbiology spectrum},
volume = {6},
number = {2},
pages = {},
pmid = {29676246},
issn = {2165-0497},
support = {R01 GM081554/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Toxins/metabolism ; Bacteriocins/metabolism ; CRISPR-Cas Systems ; Endonucleases/metabolism ; Endoribonucleases/metabolism ; Exonucleases/metabolism ; Exoribonucleases/metabolism ; Gene Expression Regulation, Bacterial ; Gram-Negative Bacteria/*enzymology ; Polyribonucleotide Nucleotidyltransferase/metabolism ; RNA Helicases/metabolism ; RNA Processing, Post-Transcriptional/*physiology ; *RNA Stability ; RNA, Bacterial/*metabolism ; RNA, Messenger/metabolism ; RNA, Transfer/metabolism ; RNA, Untranslated/metabolism ; Ribonuclease III/metabolism ; },
abstract = {Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this review, we discuss the various enzymes that control transcription, translation, and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5' and 3' termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript are matured to individual 16S, 23S, and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and nontranslated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions, Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase, as well as proteins that regulate the catalytic activity of particular RNases. Under certain stress conditions, an additional group of specialized endonucleases facilitate the cell's ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I, participate in multiple RNA processing and decay pathways.},
}

Bacterial Toxins/metabolism
Bacteriocins/metabolism
CRISPR-Cas Systems
Endonucleases/metabolism
Endoribonucleases/metabolism
Exonucleases/metabolism
Exoribonucleases/metabolism
Gene Expression Regulation, Bacterial
Gram-Negative Bacteria/*enzymology
Polyribonucleotide Nucleotidyltransferase/metabolism
RNA Helicases/metabolism
RNA Processing, Post-Transcriptional/*physiology
*RNA Stability
RNA, Bacterial/*metabolism
RNA, Messenger/metabolism
RNA, Transfer/metabolism
RNA, Untranslated/metabolism
Ribonuclease III/metabolism

@article {pmid29662124,
year = {2018},
author = {Ding, S and Diep, J and Feng, N and Ren, L and Li, B and Ooi, YS and Wang, X and Brulois, KF and Yasukawa, LL and Li, X and Kuo, CJ and Solomon, DA and Carette, JE and Greenberg, HB},
title = {STAG2 deficiency induces interferon responses via cGAS-STING pathway and restricts virus infection.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {1485},
pmid = {29662124},
issn = {2041-1723},
support = {DP5 OD021403/OD/NIH HHS/United States ; R01 AI021362/NH/NIH HHS/United States ; R56 AI021362/NH/NIH HHS/United States ; },
mesh = {Antigens, Nuclear/genetics/*immunology ; CRISPR-Cas Systems ; Caco-2 Cells ; Cell Cycle Proteins/genetics/*immunology ; Cell Nucleus/immunology/virology ; Chromosomal Proteins, Non-Histone/genetics/*immunology ; DNA Damage ; Gene Deletion ; Gene Editing ; Gene Expression Regulation ; Genome, Human ; HEK293 Cells ; HT29 Cells ; HeLa Cells ; *Host-Pathogen Interactions ; Humans ; Interferons/genetics/immunology ; Intestinal Mucosa/immunology/virology ; Janus Kinases/genetics/immunology ; Membrane Proteins/genetics/*immunology ; Nucleotidyltransferases/genetics/*immunology ; Rotavirus/growth & development/*immunology ; STAT Transcription Factors/genetics/immunology ; Signal Transduction ; Spheroids, Cellular/*immunology/virology ; },
abstract = {Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. Mechanistically, STAG2 deficiency results in spontaneous genomic DNA damage and robust interferon (IFN) expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and IFN-stimulated gene (ISG) expression broadly protects against virus infections, including RVs. Our work highlights a previously undocumented role of the cohesin complex in regulating IFN homeostasis and identifies new therapeutic avenues for manipulating the innate immunity.},
}

Antigens, Nuclear/genetics/*immunology
CRISPR-Cas Systems
Caco-2 Cells
Cell Cycle Proteins/genetics/*immunology
Cell Nucleus/immunology/virology
Chromosomal Proteins, Non-Histone/genetics/*immunology
DNA Damage
Gene Deletion
Gene Editing
Gene Expression Regulation
Genome, Human
HEK293 Cells
HT29 Cells
HeLa Cells
*Host-Pathogen Interactions
Humans
Interferons/genetics/immunology
Intestinal Mucosa/immunology/virology
Janus Kinases/genetics/immunology
Membrane Proteins/genetics/*immunology
Nucleotidyltransferases/genetics/*immunology
Rotavirus/growth & development/*immunology
STAT Transcription Factors/genetics/immunology
Signal Transduction
Spheroids, Cellular/*immunology/virology

@article {pmid29626221,
year = {2018},
author = {Soreanu, I and Hendler, A and Dahan, D and Dovrat, D and Aharoni, A},
title = {Marker-free genetic manipulations in yeast using CRISPR/CAS9 system.},
journal = {Current genetics},
volume = {64},
number = {5},
pages = {1129-1139},
pmid = {29626221},
issn = {1432-0983},
support = {2297/15//Israel Science Foundation/ ; 1340/17//Israel Science Foundation/ ; 2013358//United States-Israel Binational Science Foundation/ ; 722610//Horizon 2020/ ; },
mesh = {*CRISPR-Cas Systems ; Drug Resistance, Microbial/genetics ; Gene Editing ; Genes, Fungal ; Genetic Markers ; Promoter Regions, Genetic ; Saccharomyces cerevisiae/*genetics ; },
abstract = {The budding yeast is currently one of the major model organisms for the study of a wide variety of biological processes. Genetic manipulation of yeast involves the extensive usage of selectable markers that can lead to undesired effects. Thus, marker-free genetic manipulation in yeast is highly desirable for gene/promoter replacement and various other applications. Here we combine the power of selectable markers followed by CRISPR/CAS9 genome editing for common genetic manipulations in yeast in a marker-free manner. We demonstrate our approach for whole gene and promoter replacements and for high-efficiency operator array integration. Our approach allows the utilization of many thousands of existing strains including library strains for the generation of significant genetic changes in yeast in a marker-free and cloning-free fashion.},
}

*CRISPR-Cas Systems
Drug Resistance, Microbial/genetics
Gene Editing
Genes, Fungal
Genetic Markers
Promoter Regions, Genetic
Saccharomyces cerevisiae/*genetics

@article {pmid29623532,
year = {2018},
author = {Ben Jehuda, R and Shemer, Y and Binah, O},
title = {Genome Editing in Induced Pluripotent Stem Cells using CRISPR/Cas9.},
journal = {Stem cell reviews},
volume = {14},
number = {3},
pages = {323-336},
pmid = {29623532},
issn = {1558-6804},
mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Cell Differentiation/genetics/physiology ; Cell Line ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/*cytology/*metabolism ; },
abstract = {The development of the reprogramming technology led to generation of induced Pluripotent Stem Cells (iPSC) from a variety of somatic cells. Ever since, fast growing knowledge of different efficient protocols enabled the differentiation of these iPSCs into different cells types utilized for disease modeling. Indeed, iPSC-derived cells have been increasingly used for investigating molecular and cellular pathophysiological mechanisms underlying inherited diseases. However, a major barrier in the field of iPSC-based disease modeling relies on discriminating between the effects of the causative mutation and the genetic background of these cells. In the past decade, researchers have made great improvement in genome editing techniques, with one of the latest being CRISPR/Cas9. Using a single non-sequence specific protein combined with a small guiding RNA molecule, this state-of-the-art approach enables modifications of genes with high efficiency and accuracy. By so doing, this technique enables the generation of isogenic controls or isogenic mutated cell lines in order to focus on the pathologies caused by a specific mutation. In this article, we review the latest studies combining iPSC and CRISPR/Cas9 technologies for the investigation of the molecular and cellular mechanisms underlying inherited diseases including immunological, metabolic, hematological, neurodegenerative and cardiac diseases.},
}

Animals
CRISPR-Cas Systems/genetics/physiology
Cell Differentiation/genetics/physiology
Cell Line
Gene Editing/*methods
Humans
Induced Pluripotent Stem Cells/*cytology/*metabolism

@article {pmid29601977,
year = {2018},
author = {Quadalti, C and Brunetti, D and Lagutina, I and Duchi, R and Perota, A and Lazzari, G and Cerutti, R and Di Meo, I and Johnson, M and Bottani, E and Crociara, P and Corona, C and Grifoni, S and Tiranti, V and Fernandez-Vizarra, E and Robinson, AJ and Viscomi, C and Casalone, C and Zeviani, M and Galli, C},
title = {SURF1 knockout cloned pigs: Early onset of a severe lethal phenotype.},
journal = {Biochimica et biophysica acta. Molecular basis of disease},
volume = {1864},
number = {6 Pt A},
pages = {2131-2142},
pmid = {29601977},
issn = {0925-4439},
mesh = {Animals ; Animals, Genetically Modified ; Animals, Newborn ; Behavior, Animal ; CRISPR-Cas Systems ; Cells, Cultured ; Central Nervous System/*growth & development ; *Disease Models, Animal ; Down-Regulation ; Electron Transport Complex IV/metabolism ; Female ; Fibroblasts ; Gene Editing ; Gene Knockout Techniques ; Humans ; Jejunum/pathology ; Leigh Disease/*genetics/pathology ; Male ; Membrane Proteins/*genetics ; Mitochondria/pathology ; Mitochondrial Proteins/*genetics ; Muscle, Skeletal/cytology/pathology ; Nuclear Transfer Techniques ; Primary Cell Culture ; Sus scrofa/*genetics ; },
abstract = {Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF1-/- mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1-/- and SURF1-/+ pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1-/- pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1-/+ and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1-/- tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down-regulated in SURF1-/- skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1-/- piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics.},
}

Animals
Animals, Genetically Modified
Animals, Newborn
Behavior, Animal
CRISPR-Cas Systems
Cells, Cultured
Central Nervous System/*growth & development
*Disease Models, Animal
Down-Regulation
Electron Transport Complex IV/metabolism
Female
Fibroblasts
Gene Editing
Gene Knockout Techniques
Humans
Jejunum/pathology
Leigh Disease/*genetics/pathology
Male
Membrane Proteins/*genetics
Mitochondria/pathology
Mitochondrial Proteins/*genetics
Muscle, Skeletal/cytology/pathology
Nuclear Transfer Techniques
Primary Cell Culture
Sus scrofa/*genetics

@article {pmid29572864,
year = {2018},
author = {Andersson, M and Turesson, H and Olsson, N and Fält, AS and Ohlsson, P and Gonzalez, MN and Samuelsson, M and Hofvander, P},
title = {Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery.},
journal = {Physiologia plantarum},
volume = {164},
number = {4},
pages = {378-384},
doi = {10.1111/ppl.12731},
pmid = {29572864},
issn = {1399-3054},
support = {//Einar and Inga Nilssons Stiftelse/ ; //Lyckeby Research Foundation (Stiftelsen Stärkelsen Forskning Utveckling)/ ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Protoplasts/metabolism ; Ribonucleoproteins/*genetics ; Solanum tuberosum/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein-9 (CRISPR-Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA-free genome editing method, using delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr-RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene-free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT-RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2-3% of the regenerated shoots from the RNP-experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Protoplasts/metabolism
Ribonucleoproteins/*genetics
Solanum tuberosum/*genetics

@article {pmid29556040,
year = {2018},
author = {Charpentier, M and Khedher, AHY and Menoret, S and Brion, A and Lamribet, K and Dardillac, E and Boix, C and Perrouault, L and Tesson, L and Geny, S and De Cian, A and Itier, JM and Anegon, I and Lopez, B and Giovannangeli, C and Concordet, JP},
title = {CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {1133},
pmid = {29556040},
issn = {2041-1723},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Carrier Proteins/chemistry/genetics/*metabolism ; Cell Line ; DNA Breaks, Double-Stranded ; Enhancer Elements, Genetic ; Female ; HCT116 Cells ; HEK293 Cells ; Homologous Recombination ; Humans ; INDEL Mutation ; Induced Pluripotent Stem Cells/metabolism ; Nuclear Proteins/chemistry/genetics/*metabolism ; Oocytes/metabolism ; Phosphorylation ; Protein Multimerization ; RNA, Guide/genetics ; Rats ; Recombinant Fusion Proteins/chemistry/genetics/metabolism ; *Recombinational DNA Repair ; Transgenes ; Virus Integration/genetics ; Zygote/metabolism ; },
abstract = {In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.},
}

Animals
Base Sequence
CRISPR-Cas Systems
Carrier Proteins/chemistry/genetics/*metabolism
Cell Line
DNA Breaks, Double-Stranded
Enhancer Elements, Genetic
Female
HCT116 Cells
HEK293 Cells
Homologous Recombination
Humans
INDEL Mutation
Induced Pluripotent Stem Cells/metabolism
Nuclear Proteins/chemistry/genetics/*metabolism
Oocytes/metabolism
Phosphorylation
Protein Multimerization
RNA, Guide/genetics
Rats
Recombinant Fusion Proteins/chemistry/genetics/metabolism
*Recombinational DNA Repair
Transgenes
Virus Integration/genetics
Zygote/metabolism

@article {pmid29510160,
year = {2018},
author = {Shibata, T and Shibata, S and Ishigaki, Y and Kiyokawa, E and Ikawa, M and Singh, DP and Sasaki, H and Kubo, E},
title = {Tropomyosin 2 heterozygous knockout in mice using CRISPR-Cas9 system displays the inhibition of injury-induced epithelial-mesenchymal transition, and lens opacity.},
journal = {Mechanisms of ageing and development},
volume = {171},
number = {},
pages = {24-30},
pmid = {29510160},
issn = {1872-6216},
support = {R01 EY024589/EY/NEI NIH HHS/United States ; },
mesh = {*Aging/genetics/metabolism/pathology ; Animals ; *CRISPR-Cas Systems ; *Cataract/genetics/metabolism/pathology ; Epithelial-Mesenchymal Transition/*genetics ; Female ; *Gene Knockdown Techniques ; *Lens, Crystalline/injuries/metabolism/pathology ; Mice ; Mice, Knockout ; Tropomyosin/genetics/metabolism ; Wound Healing/*genetics ; },
abstract = {The process of epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) after cataract surgery contributes to tissue fibrosis, wound healing and lens regeneration via a mechanism not yet fully understood. Here, we show that tropomyosin 2 (Tpm2) plays a critical role in wound healing and lens aging. Posterior capsular opacification (PCO) after lens extraction surgery was accompanied by elevated expression of Tpm2. Tpm2 heterozygous knockout mice, generated via the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system showed promoted progression of cataract with age. Further, injury-induced EMT of the mouse lens epithelium, as evaluated histologically and by the expression patterns of Tpm1 and Tpm2, was attenuated in the absence of Tpm2. In conclusion, Tpm2 may be important in maintaining lens physiology and morphology. However, Tpm2 is involved in the progression of EMT during the wound healing process of mouse LECs, suggesting that inhibition of Tpm2 may suppress PCO.},
}

*Aging/genetics/metabolism/pathology
Animals
*CRISPR-Cas Systems
*Cataract/genetics/metabolism/pathology
Epithelial-Mesenchymal Transition/*genetics
Female
*Gene Knockdown Techniques
*Lens, Crystalline/injuries/metabolism/pathology
Mice
Mice, Knockout
Tropomyosin/genetics/metabolism
Wound Healing/*genetics

@article {pmid29499836,
year = {2018},
author = {Gerhards, NM and Rottenberg, S},
title = {New tools for old drugs: Functional genetic screens to optimize current chemotherapy.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {36},
number = {},
pages = {30-46},
pmid = {29499836},
issn = {1532-2084},
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Drug Resistance, Neoplasm/*genetics ; Drug Screening Assays, Antitumor/methods ; Gene Editing/methods ; Genetic Testing/methods ; High-Throughput Nucleotide Sequencing ; Humans ; Mutagenesis/genetics ; Neoplasms/*drug therapy/genetics/pathology ; Precision Medicine/*methods ; Treatment Outcome ; Tumor Cells, Cultured ; },
abstract = {Despite substantial advances in the treatment of various cancers, many patients still receive anti-cancer therapies that hardly eradicate tumor cells but inflict considerable side effects. To provide the best treatment regimen for an individual patient, a major goal in molecular oncology is to identify predictive markers for a personalized therapeutic strategy. Regarding novel targeted anti-cancer therapies, there are usually good markers available. Unfortunately, however, targeted therapies alone often result in rather short remissions and little cytotoxic effect on the cancer cells. Therefore, classical chemotherapy with frequent long remissions, cures, and a clear effect on cancer cell eradication remains a corner stone in current anti-cancer therapy. Reliable biomarkers which predict the response of tumors to classical chemotherapy are rare, in contrast to the situation for targeted therapy. For the bulk of cytotoxic therapeutic agents, including DNA-damaging drugs, drugs targeting microtubules or antimetabolites, there are still no reliable biomarkers used in the clinic to predict tumor response. To make progress in this direction, meticulous studies of classical chemotherapeutic drug action and resistance mechanisms are required. For this purpose, novel functional screening technologies have emerged as successful technologies to study chemotherapeutic drug response in a variety of models. They allow a systematic analysis of genetic contributions to a drug-responsive or -sensitive phenotype and facilitate a better understanding of the mode of action of these drugs. These functional genomic approaches are not only useful for the development of novel targeted anti-cancer drugs but may also guide the use of classical chemotherapeutic drugs by deciphering novel mechanisms influencing a tumor's drug response. Moreover, due to the advances of 3D organoid cultures from patient tumors and in vivo screens in mice, these genetic screens can be applied using conditions that are more representative of the clinical setting. Patient-derived 3D organoid lines furthermore allow the characterization of the "essentialome", the specific set of genes required for survival of these cells, of an individual tumor, which could be monitored over the course of treatment and help understanding how drug resistance evolves in clinical tumors. Thus, we expect that these functional screens will enable the discovery of novel cancer-specific vulnerabilities, and through clinical validation, move the field of predictive biomarkers forward. This review focuses on novel advanced techniques to decipher the interplay between genetic alterations and drug response.},
}

Animals
Antineoplastic Agents/*pharmacology/therapeutic use
Biomarkers, Tumor/genetics
CRISPR-Cas Systems/genetics
Disease Models, Animal
Drug Resistance, Neoplasm/*genetics
Drug Screening Assays, Antitumor/methods
Gene Editing/methods
Genetic Testing/methods
High-Throughput Nucleotide Sequencing
Humans
Mutagenesis/genetics
Neoplasms/*drug therapy/genetics/pathology
Precision Medicine/*methods
Treatment Outcome
Tumor Cells, Cultured

@article {pmid29458735,
year = {2018},
author = {Boulad, F and Mansilla-Soto, J and Cabriolu, A and Rivière, I and Sadelain, M},
title = {Gene Therapy and Genome Editing.},
journal = {Hematology/oncology clinics of North America},
volume = {32},
number = {2},
pages = {329-342},
doi = {10.1016/j.hoc.2017.11.007},
pmid = {29458735},
issn = {1558-1977},
mesh = {CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Transfer Techniques ; *Genetic Therapy/methods ; Genetic Vectors/genetics ; Humans ; Transduction, Genetic ; beta-Globins/*genetics ; beta-Thalassemia/*genetics/*therapy ; },
abstract = {The β-thalassemias are inherited blood disorders that result from insufficient production of the β-chain of hemoglobin. More than 200 different mutations have been identified. β-Thalassemia major requires life-long transfusions. The only cure for severe β-thalassemia is to provide patients with hematopoietic stem cells. Globin gene therapy promises a curative autologous stem cell transplantation without the immunologic complications of allogeneic transplantation. The future directions of gene therapy include enhancement of lentiviral vector-based approaches, fine tuning of the conditioning regimen, and the design of safer vectors. Progress in genetic engineering bodes well for finding a cure for severe globin disorders.},
}

CRISPR-Cas Systems
*Gene Editing/methods
Gene Transfer Techniques
*Genetic Therapy/methods
Genetic Vectors/genetics
Humans
Transduction, Genetic
beta-Globins/*genetics
beta-Thalassemia/*genetics/*therapy

@article {pmid29439141,
year = {2018},
author = {Song, Y and Xu, Y and Liang, M and Zhang, Y and Chen, M and Deng, J and Li, Z},
title = {CRISPR/Cas9-mediated mosaic mutation of SRY gene induces hermaphroditism in rabbits.},
journal = {Bioscience reports},
volume = {38},
number = {2},
pages = {},
pmid = {29439141},
issn = {1573-4935},
mesh = {Animals ; *CRISPR-Cas Systems ; *Chimerism ; Disease Models, Animal ; Female ; Male ; *Mutation ; Ovotesticular Disorders of Sex Development/*genetics/metabolism/pathology ; Rabbits ; Sex-Determining Region Y Protein/*genetics/metabolism ; },
abstract = {Hermaphroditism is a rare disorder that affects sexual development, resulting in individuals with both male and female sexual organs. Hermaphroditism is caused by anomalies in genes regulating sex determination, gonad development, or expression of hormones and their receptors during embryonic development during sexual differentiation. SRY is a sex-determination gene on the Y chromosome that is responsible for initiating male sex determination in mammals. In this study, we introduced CRISPR/Cas9-mediated mutations in the high-mobility-group (HMG) region of the rabbit SRY As expected, SRY-mutant chimeric rabbits were diagnosed with hermaphroditism, characterized by possessing ovotestis, testis, ovary and uterus simultaneously. Histopathology analysis revealed that the testicular tissue was immature and lacked spermatogenic cells, while the ovarian portion appeared normal and displayed follicles at different stages. This is the first report of a rabbit hermaphroditism model generated by the CRISPR/Cas9 system. This novel rabbit model could advance our understanding of the pathogenesis of hermaphroditism, and identify novel therapies for human clinical treatment of hermaphroditism.},
}

Animals
*CRISPR-Cas Systems
*Chimerism
Disease Models, Animal
Female
Male
*Mutation
Ovotesticular Disorders of Sex Development/*genetics/metabolism/pathology
Rabbits
Sex-Determining Region Y Protein/*genetics/metabolism

@article {pmid29423928,
year = {2018},
author = {Sakuma, T and Mochida, K and Nakade, S and Ezure, T and Minagawa, S and Yamamoto, T},
title = {Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {23},
number = {4},
pages = {255-263},
doi = {10.1111/gtc.12569},
pmid = {29423928},
issn = {1365-2443},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cells, Cultured ; Clone Cells/*metabolism ; *Gene Editing ; *Genetic Heterogeneity ; HCT116 Cells ; Humans ; Hypoxanthine Phosphoribosyltransferase/*genetics ; Mutagenesis ; RNA, Guide ; Ribonucleoproteins/*genetics ; Single-Cell Analysis ; },
abstract = {Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures.},
}

Base Sequence
CRISPR-Cas Systems
Cells, Cultured
Clone Cells/*metabolism
*Gene Editing
*Genetic Heterogeneity
HCT116 Cells
Humans
Hypoxanthine Phosphoribosyltransferase/*genetics
Mutagenesis
RNA, Guide
Ribonucleoproteins/*genetics
Single-Cell Analysis

@article {pmid29391533,
year = {2018},
author = {Guo, Q and Mintier, G and Ma-Edmonds, M and Storton, D and Wang, X and Xiao, X and Kienzle, B and Zhao, D and Feder, JN},
title = {'Cold shock' increases the frequency of homology directed repair gene editing in induced pluripotent stem cells.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {2080},
pmid = {29391533},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Cold Temperature ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; *Recombinational DNA Repair ; },
abstract = {Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20-40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1-20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by 'cold shocking' cells at 32 °C for 24-48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or 'perfect HDR', as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated ('partial HDR'). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing 'perfect HDR' compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.},
}

CRISPR-Cas Systems
Cold Temperature
Gene Editing/*methods
HEK293 Cells
Humans
Induced Pluripotent Stem Cells/*metabolism
*Recombinational DNA Repair

@article {pmid29379029,
year = {2018},
author = {Khoshnejad, M and Brenner, JS and Motley, W and Parhiz, H and Greineder, CF and Villa, CH and Marcos-Contreras, OA and Tsourkas, A and Muzykantov, VR},
title = {Molecular engineering of antibodies for site-specific covalent conjugation using CRISPR/Cas9.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {1760},
pmid = {29379029},
issn = {2045-2322},
support = {T32 HL007954/HL/NHLBI NIH HHS/United States ; R01 HL125462/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Antibodies, Monoclonal/*genetics ; Antigens/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cysteine/genetics ; Humans ; Immunoconjugates/*genetics ; Mice ; Rats ; Reproducibility of Results ; },
abstract = {Site-specific modification of antibodies has become a critical aspect in the development of next-generation immunoconjugates meeting criteria of clinically acceptable homogeneity, reproducibility, efficacy, ease of manufacturability, and cost-effectiveness. Using CRISPR/Cas9 genomic editing, we developed a simple and novel approach to produce site-specifically modified antibodies. A sortase tag was genetically incorporated into the C-terminal end of the third immunoglobulin heavy chain constant region (CH3) within a hybridoma cell line to manufacture antibodies capable of site-specific conjugation. This enabled an effective enzymatic site-controlled conjugation of fluorescent and radioactive cargoes to a genetically tagged mAb without impairment of antigen binding activity. After injection in mice, these immunoconjugates showed almost doubled specific targeting in the lung vs. chemically conjugated maternal mAb, and concomitant reduction in uptake in the liver and spleen. The approach outlined in this work provides a facile method for the development of more homogeneous, reproducible, effective, and scalable antibody conjugates for use as therapeutic and diagnostic tools.},
}

Animals
Antibodies, Monoclonal/*genetics
Antigens/genetics
CRISPR-Cas Systems/*genetics
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cysteine/genetics
Humans
Immunoconjugates/*genetics
Mice
Rats
Reproducibility of Results

@article {pmid29358698,
year = {2018},
author = {Jiang, W and Liu, L and Chang, Q and Xing, F and Ma, Z and Fang, Z and Zhou, J and Fu, L and Wang, H and Huang, X and Chen, X and Li, Y and Li, S},
title = {Production of Wilson Disease Model Rabbits with Homology-Directed Precision Point Mutations in the ATP7B Gene Using the CRISPR/Cas9 System.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {1332},
pmid = {29358698},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Copper/analysis ; Copper-transporting ATPases/*genetics ; DNA, Single-Stranded/administration & dosage/pharmacology ; *Disease Models, Animal ; Hepatolenticular Degeneration/*genetics ; Humans ; Liver/chemistry ; Microinjections ; Mutagenesis, Site-Directed ; Oligodeoxyribonucleotides/*administration & dosage/pharmacology ; *Point Mutation ; Rabbits ; Zygote/chemistry/growth & development ; },
abstract = {CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.},
}

Animals
CRISPR-Cas Systems
Copper/analysis
Copper-transporting ATPases/*genetics
DNA, Single-Stranded/administration & dosage/pharmacology
*Disease Models, Animal
Hepatolenticular Degeneration/*genetics
Humans
Liver/chemistry
Microinjections
Mutagenesis, Site-Directed
Oligodeoxyribonucleotides/*administration & dosage/pharmacology
*Point Mutation
Rabbits
Zygote/chemistry/growth & development

@article {pmid29247817,
year = {2018},
author = {Kim, H and Kim, JW and Kim, SJ and Choi, YJ and Kim, DS and Park, HJ},
title = {Generation of a PXR reporter human induced pluripotent stem cell line (PXR-mCherry hiPSC) using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {26},
number = {},
pages = {72-75},
doi = {10.1016/j.scr.2017.12.001},
pmid = {29247817},
issn = {1876-7753},
mesh = {*CRISPR-Cas Systems ; *Cell Differentiation ; Cell Line ; Cell Proliferation ; Cells, Cultured ; *Genes, Reporter ; Hepatocytes/*cytology/metabolism ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Peroxisome-Targeting Signal 1 Receptor/antagonists & inhibitors/*genetics ; },
abstract = {Pregnane X receptor (PXR) is a key nuclear receptor that mediates drug metabolism and stimulates hepatocyte proliferation. However, the lack of PXR expression in human pluripotent stem cell-derived hepatocytes limits their application for drug screening and toxicity testing. Here, we generated a PXR-mCherry reporter human induced pluripotent stem cell (hiPSC) line using the CRISPR/Cas9 system. PXR-mCherry hiPSCs were pluripotent and had differentiation potential and a normal karyotype. This cell line is an important tool for identifying factors that increase PXR-mediated drug metabolism and hepatocyte proliferation.},
}

*CRISPR-Cas Systems
*Cell Differentiation
Cell Line
Cell Proliferation
Cells, Cultured
*Genes, Reporter
Hepatocytes/*cytology/metabolism
Humans
Induced Pluripotent Stem Cells/*cytology/metabolism
Peroxisome-Targeting Signal 1 Receptor/antagonists & inhibitors/*genetics

@article {pmid29247816,
year = {2018},
author = {Yuan, F and Guo, D and Liu, Y and Xu, Y and Gao, G and Wu, Y and Yang, F and Ke, X and Lai, K and Wei, H and Li, YX},
title = {Generation of an ASS1 heterozygous knockout human embryonic stem cell line, WAe001-A-13, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {26},
number = {},
pages = {67-71},
doi = {10.1016/j.scr.2017.11.007},
pmid = {29247816},
issn = {1876-7753},
mesh = {Argininosuccinate Lyase/antagonists & inhibitors/*genetics/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Cell Line ; Heterozygote ; Human Embryonic Stem Cells/*cytology/physiology ; Humans ; Mutation ; Phenotype ; },
abstract = {The ASS1 gene encodes argininosuccinate synthetase-1, a cytosolic enzyme with a critical role in the urea cycle. Mutations are found in all ASS1 exons and cause the autosomal recessive disorder citrullinemia. Using CRISPR/Cas9-editing, we established the WAe001-A-13 cell line, which was heterozygous for an ASS1 mutation, from the human embryonic stem cell line H1. The WAe001-A-13 cell line maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype.},
}

Argininosuccinate Lyase/antagonists & inhibitors/*genetics/metabolism
Base Sequence
*CRISPR-Cas Systems
Cell Line
Heterozygote
Human Embryonic Stem Cells/*cytology/physiology
Humans
Mutation
Phenotype

@article {pmid29238966,
year = {2018},
author = {Sim, SW and Park, TS and Kim, SJ and Park, BC and Weinstein, DA and Lee, YM and Jun, HS},
title = {Aberrant proliferation and differentiation of glycogen storage disease type Ib mesenchymal stem cells.},
journal = {FEBS letters},
volume = {592},
number = {2},
pages = {162-171},
doi = {10.1002/1873-3468.12939},
pmid = {29238966},
issn = {1873-3468},
mesh = {*Adipogenesis ; Adipose Tissue/*cytology/metabolism ; Antiporters/*genetics ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclooxygenase 2/metabolism ; Dinoprostone/metabolism ; Glycogen Storage Disease Type I/*genetics ; Glycolysis ; Humans ; Mesenchymal Stem Cells/*cytology/metabolism ; Monosaccharide Transport Proteins/*genetics ; *Osteogenesis ; Phenotype ; Single-Cell Analysis ; },
abstract = {Glycogen storage disease type Ib (GSD-Ib) is caused by mutations of the glucose-6-phosphate transporter (G6PT) and characterized by disrupted glucose homeostasis, neutropenia, and neutrophil dysfunction. To investigate the role of G6PT in human adipose-derived mesenchymal stem cells (hMSCs), the G6PT gene was mutated by CRISPR/Cas9 technology and single cell-derived G6PT-/- hMSCs were established. G6PT-/- hMSCs have significantly increased cell proliferation but impaired adipogenesis and osteogenesis. These phenotypes are associated with two mechanisms: i) metabolic reprogramming in G6PT-/- hMSCs causing a metabolic shift toward glycolysis rather than oxidative phosphorylation and ii) increased cyclooxygenase-2-derived prostaglandin E2 secretion in G6PT-/- hMSCs. This study demonstrates that G6PT is essential for proliferation and differentiation of MSCs, providing important insights into the GSD-Ib phenotypes.},
}

*Adipogenesis
Adipose Tissue/*cytology/metabolism
Antiporters/*genetics
CRISPR-Cas Systems
Cell Differentiation
Cell Proliferation
Cells, Cultured
Cyclooxygenase 2/metabolism
Dinoprostone/metabolism
Glycogen Storage Disease Type I/*genetics
Glycolysis
Humans
Mesenchymal Stem Cells/*cytology/metabolism
Monosaccharide Transport Proteins/*genetics
*Osteogenesis
Phenotype
Single-Cell Analysis

@article {pmid29205102,
year = {2018},
author = {Markossian, S and Guyot, R and Richard, S and Teixeira, M and Aguilera, N and Bouchet, M and Plateroti, M and Guan, W and Gauthier, K and Aubert, D and Flamant, F},
title = {CRISPR/Cas9 Editing of the Mouse Thra Gene Produces Models with Variable Resistance to Thyroid Hormone.},
journal = {Thyroid : official journal of the American Thyroid Association},
volume = {28},
number = {1},
pages = {139-150},
doi = {10.1089/thy.2017.0389},
pmid = {29205102},
issn = {1557-9077},
mesh = {Animals ; CRISPR-Cas Systems ; Genes, erbA/*genetics ; Mice ; Mutation ; Phenotype ; Thyroid Hormone Resistance Syndrome/*genetics ; },
abstract = {BACKGROUND: Resistance to thyroid hormone due to THRA mutations (RTHα) is a recently discovered genetic disease, displaying important variability in its clinical presentation. The mutations alter the function of TRα1, one of the two nuclear receptors for thyroid hormone.

METHODS: The aim of this study was to understand the relationship between specific THRA mutations and phenotype. CRISPR/Cas9 genome editing was used to generate five new mouse models of RTHα, with frameshift or missense mutations.

RESULTS: Like human patients, mutant mice displayed a hypothyroid-like phenotype, with altered development. Phenotype severity varied between the different mouse models, mainly depending on the ability of the mutant receptor to interact with transcription corepressor in the presence of thyroid hormone.

CONCLUSION: The present mutant mice represent highly relevant models for the human genetic disease which will be useful for future investigations.},
}

Animals
CRISPR-Cas Systems
Genes, erbA/*genetics
Mice
Mutation
Phenotype
Thyroid Hormone Resistance Syndrome/*genetics

@article {pmid29179920,
year = {2018},
author = {Gammage, PA and Moraes, CT and Minczuk, M},
title = {Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized.},
journal = {Trends in genetics : TIG},
volume = {34},
number = {2},
pages = {101-110},
pmid = {29179920},
issn = {0168-9525},
support = {MC_U105697135//Medical Research Council/United Kingdom ; },
mesh = {Animals ; Biolistics/methods ; Biological Transport ; *CRISPR-Cas Systems ; DNA, Mitochondrial/*genetics/metabolism ; Dependovirus/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/*methods ; Genetic Vectors/chemistry/metabolism ; *Genome, Mitochondrial ; Mammals ; Mitochondria/*genetics/metabolism ; Polyribonucleotide Nucleotidyltransferase/genetics/metabolism ; RNA, Guide/genetics/metabolism ; Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Transcription Factors/genetics/metabolism ; },
abstract = {In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial.},
}

Animals
Biolistics/methods
Biological Transport
*CRISPR-Cas Systems
DNA, Mitochondrial/*genetics/metabolism
Dependovirus/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism
Gene Editing/*methods
Genetic Vectors/chemistry/metabolism
*Genome, Mitochondrial
Mammals
Mitochondria/*genetics/metabolism
Polyribonucleotide Nucleotidyltransferase/genetics/metabolism
RNA, Guide/genetics/metabolism
Transcription Activator-Like Effector Nucleases/genetics/metabolism
Transcription Factors/genetics/metabolism

@article {pmid28853250,
year = {2017},
author = {Zhou, W and Guo, R and Deng, M and Wang, F and Zhang, Y},
title = {[RS-1 enhanced the efficiency of CRISPR-Cas9 mediated knock-in of human lactoferrin].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {33},
number = {8},
pages = {1224-1234},
doi = {10.13345/j.cjb.170105},
pmid = {28853250},
issn = {1000-3061},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Fibroblasts ; *Gene Knock-In Techniques ; Genetic Vectors ; Goats ; Humans ; Lactoferrin/*genetics ; Lactoglobulins/*genetics ; Transfection ; },
abstract = {This study aims to knock out the goat β-lactoglobulin (BLG) gene using CRISPR-Cas9 system and knock in human lactoferrin (hLF) at the BLG locus, and further study the effect of RAD51 stimulatory compound (RS-1) on homologous recombination efficiency. First, we designed an sgRNA targeting the first exon of goat BLG gene and constructed a co-expression vector pCas9-sgBLG. This sgRNA vector was then transfected into goat ear fibroblasts (GEFs), and the target region was examined by T7EN1 assay and sequencing. Second, we constructed a targeting vector pBHA-hLF-NIE including NEO and EGFP genes based on BLG gene locus. This targeting vector together with pCas9-sgBLG expression vector was co-transfected into GEFs. Transfected cells were then treated with 0, 5, 10 and 20 μmol/L RS-1 for 72 h to analyse the EGFP expression efficiency. Next, we used 800 μg/mL G418 to screen G418-resistent cell clones, and studied hLF site-specific knock-in cell clones by PCR and sequencing. The editing efficiency of sgBLG was between 25% and 31%. The EGFP expression efficiency indicated that the gene knock-in efficiency was improved by RS-1 in a dose-dependent manner, which could reach 3.5-fold compared to the control group. The percentage of positive cells with hLF knock-in was increased to 32.61% when 10 μmol/L RS-1 was used. However, when the concentration of RS-1 increased to 20 μmol/L, the percentage of positive cells decreased to 22.22% and resulted in an increase of senescent cell clone number. These results suggested that hLF knock-in and BLG knock-out in GEFs were achieved by using CRISPR/Cas9 system, and optimum concentration of RS-1 could improve knock-in efficiency, which provides a reference for efficiently obtaining gene knock-in cells using CRISPR/Cas9 in the future.},
}

Animals
*CRISPR-Cas Systems
Cells, Cultured
Fibroblasts
*Gene Knock-In Techniques
Genetic Vectors
Goats
Humans
Lactoferrin/*genetics
Lactoglobulins/*genetics
Transfection

@article {pmid28703221,
year = {2017},
author = {Morgan, SL and Mariano, NC and Bermudez, A and Arruda, NL and Wu, F and Luo, Y and Shankar, G and Jia, L and Chen, H and Hu, JF and Hoffman, AR and Huang, CC and Pitteri, SJ and Wang, KC},
title = {Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15993},
pmid = {28703221},
issn = {2041-1723},
support = {I01 BX002905/BX/BLRD VA/United States ; T32 CA009302/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Chromatin Assembly and Disassembly ; DEAD-box RNA Helicases/metabolism ; HEK293 Cells ; Humans ; K562 Cells ; Promoter Regions, Genetic ; beta-Globins/genetics ; },
abstract = {Chromatin looping is key to gene regulation, yet no broadly applicable methods to selectively modify chromatin loops have been described. We have engineered a method for chromatin loop reorganization using CRISPR-dCas9 (CLOuD9) to selectively and reversibly establish chromatin loops. We demonstrate the power of this technology to selectively modulate gene expression at targeted loci.},
}

*CRISPR-Cas Systems
*Chromatin Assembly and Disassembly
DEAD-box RNA Helicases/metabolism
HEK293 Cells
Humans
K562 Cells
Promoter Regions, Genetic
beta-Globins/genetics

@article {pmid28695888,
year = {2017},
author = {Cook, PJ and Thomas, R and Kannan, R and de Leon, ES and Drilon, A and Rosenblum, MK and Scaltriti, M and Benezra, R and Ventura, A},
title = {Somatic chromosomal engineering identifies BCAN-NTRK1 as a potent glioma driver and therapeutic target.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15987},
pmid = {28695888},
issn = {2041-1723},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; T32 CA160001/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Benzamides/pharmacology/*therapeutic use ; Brevican/*genetics ; CRISPR-Cas Systems ; Drug Screening Assays, Antitumor ; Feasibility Studies ; Female ; Gene Editing ; Glioma/drug therapy/*genetics ; Humans ; Indazoles/pharmacology/*therapeutic use ; Mice, Inbred C57BL ; Mice, Nude ; Neoplasms, Experimental ; *Oncogene Fusion ; Primary Cell Culture ; Receptor, trkA/antagonists & inhibitors/*genetics ; },
abstract = {The widespread application of high-throughput sequencing methods is resulting in the identification of a rapidly growing number of novel gene fusions caused by tumour-specific chromosomal rearrangements, whose oncogenic potential remains unknown. Here we describe a strategy that builds upon recent advances in genome editing and combines ex vivo and in vivo chromosomal engineering to rapidly and effectively interrogate the oncogenic potential of genomic rearrangements identified in human brain cancers. We show that one such rearrangement, an microdeletion resulting in a fusion between Brevican (BCAN) and Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1), is a potent oncogenic driver of high-grade gliomas and confers sensitivity to the experimental TRK inhibitor entrectinib. This work demonstrates that BCAN-NTRK1 is a bona fide human glioma driver and describes a general strategy to define the oncogenic potential of novel glioma-associated genomic rearrangements and to generate accurate preclinical models of this lethal human cancer.},
}

Animals
Benzamides/pharmacology/*therapeutic use
Brevican/*genetics
CRISPR-Cas Systems
Drug Screening Assays, Antitumor
Feasibility Studies
Female
Gene Editing
Glioma/drug therapy/*genetics
Humans
Indazoles/pharmacology/*therapeutic use
Mice, Inbred C57BL
Mice, Nude
Neoplasms, Experimental
*Oncogene Fusion
Primary Cell Culture
Receptor, trkA/antagonists & inhibitors/*genetics

@article {pmid28691711,
year = {2017},
author = {Huang, J and Chen, M and Whitley, MJ and Kuo, HC and Xu, ES and Walens, A and Mowery, YM and Van Mater, D and Eward, WC and Cardona, DM and Luo, L and Ma, Y and Lopez, OM and Nelson, CE and Robinson-Hamm, JN and Reddy, A and Dave, SS and Gersbach, CA and Dodd, RD and Kirsch, DG},
title = {Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15999},
pmid = {28691711},
issn = {2041-1723},
support = {F30 CA206424/CA/NCI NIH HHS/United States ; P30 CA086862/CA/NCI NIH HHS/United States ; R35 CA197616/CA/NCI NIH HHS/United States ; T32 GM007171/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Electroporation ; Gene Editing/methods ; *Integrases ; Male ; Mice ; Mice, Nude ; Mutation ; NIH 3T3 Cells ; Neurilemmoma/genetics/pathology ; Sarcoma, Experimental/*genetics/pathology ; },
abstract = {Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers.},
}

Animals
*CRISPR-Cas Systems
Electroporation
Gene Editing/methods
*Integrases
Male
Mice
Mice, Nude
Mutation
NIH 3T3 Cells
Neurilemmoma/genetics/pathology
Sarcoma, Experimental/*genetics/pathology

@article {pmid28605576,
year = {2018},
author = {Hu, X and Meng, X and Liu, Q and Li, J and Wang, K},
title = {Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice.},
journal = {Plant biotechnology journal},
volume = {16},
number = {1},
pages = {292-297},
pmid = {28605576},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Oryza/*genetics ; Streptococcus pyogenes/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR-Cas9-VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Oryza/*genetics
Streptococcus pyogenes/genetics

@article {pmid28569755,
year = {2017},
author = {Quinn, ME and Goh, Q and Kurosaka, M and Gamage, DG and Petrany, MJ and Prasad, V and Millay, DP},
title = {Myomerger induces fusion of non-fusogenic cells and is required for skeletal muscle development.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15665},
pmid = {28569755},
issn = {2041-1723},
support = {R01 AR068286/AR/NIAMS NIH HHS/United States ; T32 HL007752/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Communication ; Cell Differentiation ; *Cell Fusion ; Computational Biology ; Female ; Fibroblasts/cytology ; Male ; Membrane Proteins/*physiology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; *Muscle Development ; Muscle Fibers, Skeletal/*physiology ; Muscle Proteins/*physiology ; Myoblasts/cytology ; NIH 3T3 Cells ; Oligonucleotide Array Sequence Analysis ; },
abstract = {Despite the importance of cell fusion for mammalian development and physiology, the factors critical for this process remain to be fully defined, which has severely limited our ability to reconstitute cell fusion. Myomaker (Tmem8c) is a muscle-specific protein required for myoblast fusion. Expression of myomaker in fibroblasts drives their fusion with myoblasts, but not with other myomaker-expressing fibroblasts, highlighting the requirement of additional myoblast-derived factors for fusion. Here we show that Gm7325, which we name myomerger, induces the fusion of myomaker-expressing fibroblasts. Thus, myomaker and myomerger together confer fusogenic activity to otherwise non-fusogenic cells. Myomerger is skeletal muscle-specific and genetic deletion in mice results in a paucity of muscle fibres demonstrating its requirement for normal muscle formation. Myomerger deficient myocytes differentiate and harbour organized sarcomeres but are fusion-incompetent. Our findings identify myomerger as a fundamental myoblast fusion protein and establish a system that begins to reconstitute mammalian cell fusion.},
}

Animals
CRISPR-Cas Systems
Cell Communication
Cell Differentiation
*Cell Fusion
Computational Biology
Female
Fibroblasts/cytology
Male
Membrane Proteins/*physiology
Mice
Mice, Inbred C57BL
Mice, Knockout
*Muscle Development
Muscle Fibers, Skeletal/*physiology
Muscle Proteins/*physiology
Myoblasts/cytology
NIH 3T3 Cells
Oligonucleotide Array Sequence Analysis

@article {pmid28569745,
year = {2017},
author = {Zhang, Q and Vashisht, AA and O'Rourke, J and Corbel, SY and Moran, R and Romero, A and Miraglia, L and Zhang, J and Durrant, E and Schmedt, C and Sampath, SC and Sampath, SC},
title = {The microprotein Minion controls cell fusion and muscle formation.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15664},
pmid = {28569745},
issn = {2041-1723},
mesh = {3' Untranslated Regions ; Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cytoskeleton/*physiology ; Female ; Genotype ; Lung/embryology ; Male ; Mass Spectrometry ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Muscle, Skeletal/*physiology ; Myoblasts/cytology ; *Open Reading Frames ; Regeneration ; Stem Cells ; },
abstract = {Although recent evidence has pointed to the existence of small open reading frame (smORF)-encoded microproteins in mammals, their function remains to be determined. Skeletal muscle development requires fusion of mononuclear progenitors to form multinucleated myotubes, a critical but poorly understood process. Here we report the identification of Minion (microprotein inducer of fusion), a smORF encoding an essential skeletal muscle specific microprotein. Myogenic progenitors lacking Minion differentiate normally but fail to form syncytial myotubes, and Minion-deficient mice die perinatally and demonstrate a marked reduction in fused muscle fibres. The fusogenic activity of Minion is conserved in the human orthologue, and co-expression of Minion and the transmembrane protein Myomaker is sufficient to induce cellular fusion accompanied by rapid cytoskeletal rearrangement, even in non-muscle cells. These findings establish Minion as a novel microprotein required for muscle development, and define a two-component programme for the induction of mammalian cell fusion. Moreover, these data also significantly expand the known functions of smORF-encoded microproteins.},
}

3' Untranslated Regions
Animals
CRISPR-Cas Systems
Cell Differentiation
Cytoskeleton/*physiology
Female
Genotype
Lung/embryology
Male
Mass Spectrometry
Mice
Mice, Inbred C3H
Mice, Inbred C57BL
Muscle, Skeletal/*physiology
Myoblasts/cytology
*Open Reading Frames
Regeneration
Stem Cells

@article {pmid28533524,
year = {2017},
author = {Metzakopian, E and Strong, A and Iyer, V and Hodgkins, A and Tzelepis, K and Antunes, L and Friedrich, MJ and Kang, Q and Davidson, T and Lamberth, J and Hoffmann, C and Davis, GD and Vassiliou, GS and Skarnes, WC and Bradley, A},
title = {Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {2244},
pmid = {28533524},
issn = {2045-2322},
support = {//Wellcome Trust/United Kingdom ; MC_PC_12009//Medical Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; GPI-Linked Proteins/metabolism ; *Gene Editing/methods ; Gene Library ; Genetic Vectors ; *Genome-Wide Association Study/methods ; Humans ; Lentivirus/genetics ; Mice ; Phenotype ; RNA, Guide ; Signal Transduction ; },
abstract = {CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.},
}

Animals
*CRISPR-Cas Systems
GPI-Linked Proteins/metabolism
*Gene Editing/methods
Gene Library
Genetic Vectors
*Genome-Wide Association Study/methods
Humans
Lentivirus/genetics
Mice
Phenotype
RNA, Guide
Signal Transduction

@article {pmid28522803,
year = {2017},
author = {Satomura, A and Nishioka, R and Mori, H and Sato, K and Kuroda, K and Ueda, M},
title = {Precise genome-wide base editing by the CRISPR Nickase system in yeast.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {2095},
pmid = {28522803},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; DNA Repair ; Deoxyribonuclease I/genetics/*metabolism ; Gene Editing/*methods ; Genome, Fungal ; Saccharomyces cerevisiae/*genetics ; },
abstract = {The CRISPR/Cas9 system has been applied to efficient genome editing in many eukaryotic cells. However, the bases that can be edited by this system have been limited to those within the protospacer adjacent motif (PAM) and guide RNA-targeting sequences. In this study, we developed a genome-wide base editing technology, "CRISPR Nickase system" that utilizes a single Cas9 nickase. This system was free from the limitation of editable bases that was observed in the CRISPR/Cas9 system, and was able to precisely edit bases up to 53 bp from the nicking site. In addition, this system showed no off-target editing, in contrast to the CRISPR/Cas9 system. Coupling the CRISPR Nickase system with yeast gap repair cloning enabled the construction of yeast mutants within only five days. The CRISPR Nickase system provides a versatile and powerful technology for rapid, site-specific, and precise base editing in yeast.},
}

*CRISPR-Cas Systems
DNA Repair
Deoxyribonuclease I/genetics/*metabolism
Gene Editing/*methods
Genome, Fungal
Saccharomyces cerevisiae/*genetics

@article {pmid28509421,
year = {2018},
author = {Cai, Y and Chen, L and Liu, X and Guo, C and Sun, S and Wu, C and Jiang, B and Han, T and Hou, W},
title = {CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.},
journal = {Plant biotechnology journal},
volume = {16},
number = {1},
pages = {176-185},
pmid = {28509421},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/*genetics/physiology ; Flowers/genetics/*metabolism/*physiology ; Gene Editing ; Genome, Plant/genetics ; Mutagenesis/genetics/physiology ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism/physiology ; Soybeans/genetics/*metabolism/*physiology ; },
abstract = {Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens-mediated transformation. Site-directed mutations were observed at all targeted sites by DNA sequencing analysis. T1-generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1-bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58', E116°20'). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long-day and short-day conditions. We identified some 'transgene-clean' soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These 'transgene-clean' mutants of GmFT2a may provide materials for more in-depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction.},
}

CRISPR-Cas Systems/*genetics/physiology
Flowers/genetics/*metabolism/*physiology
Gene Editing
Genome, Plant/genetics
Mutagenesis/genetics/physiology
Plant Proteins/genetics/metabolism
Plants, Genetically Modified/genetics/metabolism/physiology
Soybeans/genetics/*metabolism/*physiology

@article {pmid28499063,
year = {2018},
author = {Wang, P and Zhang, J and Sun, L and Ma, Y and Xu, J and Liang, S and Deng, J and Tan, J and Zhang, Q and Tu, L and Daniell, H and Jin, S and Zhang, X},
title = {High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system.},
journal = {Plant biotechnology journal},
volume = {16},
number = {1},
pages = {137-150},
pmid = {28499063},
issn = {1467-7652},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Gossypium/*genetics ; Plants, Genetically Modified/*genetics ; Xylem/genetics ; },
abstract = {Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene-specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site-specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2-edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7-100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode-based high-throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1-edited T0 plants and it matched well with Sanger sequencing results. No off-target editing was detected at the potential off-target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Genome, Plant/*genetics
Gossypium/*genetics
Plants, Genetically Modified/*genetics
Xylem/genetics

@article {pmid28469192,
year = {2017},
author = {Zou, Z and Huang, K and Wei, Y and Chen, H and Liu, Z and Jin, M},
title = {Construction of a highly efficient CRISPR/Cas9-mediated duck enteritis virus-based vaccine against H5N1 avian influenza virus and duck Tembusu virus infection.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {1478},
pmid = {28469192},
issn = {2045-2322},
mesh = {Animals ; Antibodies, Neutralizing/biosynthesis ; Antibodies, Viral/*biosynthesis ; *CRISPR-Cas Systems ; Cross Protection ; Ducks ; Flavivirus/genetics/immunology/pathogenicity ; Flavivirus Infections/immunology/mortality/*prevention & control/virology ; Gene Editing/methods ; Hemagglutinin Glycoproteins, Influenza Virus/genetics/immunology ; Influenza A Virus, H5N1 Subtype/genetics/immunology/pathogenicity ; Influenza in Birds/immunology/mortality/*prevention & control/virology ; Mardivirus/genetics/immunology/pathogenicity ; Marek Disease/immunology/mortality/*prevention & control/virology ; Poultry Diseases/immunology/mortality/*prevention & control/virology ; Survival Analysis ; Vaccination ; Vaccines, Synthetic ; Viral Envelope Proteins/genetics/immunology ; Viral Vaccines/administration & dosage/biosynthesis/*genetics ; },
abstract = {Duck enteritis virus (DEV), duck tembusu virus (DTMUV), and highly pathogenic avian influenza virus (HPAIV) H5N1 are the most important viral pathogens in ducks, as they cause significant economic losses in the duck industry. Development of a novel vaccine simultaneously effective against these three viruses is the most economical method for reducing losses. In the present study, by utilizing a clustered regularly interspaced short palindromic repeats (CRISPR)/associated 9 (Cas9)-mediated gene editing strategy, we efficiently generated DEV recombinants (C-KCE-HA/PrM-E) that simultaneously encode the hemagglutinin (HA) gene of HPAIV H5N1 and pre-membrane proteins (PrM), as well as the envelope glycoprotein (E) gene of DTMUV, and its potential as a trivalent vaccine was also evaluated. Ducks immunized with C-KCE-HA/PrM-E enhanced both humoral and cell-mediated immune responses to H5N1 and DTMUV. Importantly, a single-dose of C-KCE-HA/PrM-E conferred solid protection against virulent H5N1, DTMUV, and DEV challenges. In conclusion, these results demonstrated for the first time that the CRISPR/Cas9 system can be applied for modification of the DEV genome rapidly and efficiently, and that recombinant C-KCE-HA/PrM-E can serve as a potential candidate trivalent vaccine to prevent H5N1, DTMUV, and DEV infections in ducks.},
}

Animals
Antibodies, Neutralizing/biosynthesis
Antibodies, Viral/*biosynthesis
*CRISPR-Cas Systems
Cross Protection
Ducks
Flavivirus/genetics/immunology/pathogenicity
Flavivirus Infections/immunology/mortality/*prevention & control/virology
Gene Editing/methods
Hemagglutinin Glycoproteins, Influenza Virus/genetics/immunology
Influenza A Virus, H5N1 Subtype/genetics/immunology/pathogenicity
Influenza in Birds/immunology/mortality/*prevention & control/virology
Mardivirus/genetics/immunology/pathogenicity
Marek Disease/immunology/mortality/*prevention & control/virology
Poultry Diseases/immunology/mortality/*prevention & control/virology
Survival Analysis
Vaccination
Vaccines, Synthetic
Viral Envelope Proteins/genetics/immunology
Viral Vaccines/administration & dosage/biosynthesis/*genetics

@article {pmid28469159,
year = {2017},
author = {Wu, J and Zhang, X and Zhu, Y and Tan, Q and He, J and Dong, M},
title = {Rational modular design of metabolic network for efficient production of plant polyphenol pinosylvin.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {1459},
pmid = {28469159},
issn = {2045-2322},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cinnamates/metabolism ; Escherichia coli/*enzymology/genetics ; Escherichia coli Proteins/genetics/*metabolism ; Gene Editing/methods ; Glucose/metabolism ; Malonyl Coenzyme A/metabolism ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/*genetics ; Pinus sylvestris/chemistry ; Plasmids/chemistry/metabolism ; Polyphenols/*biosynthesis/isolation & purification ; Stilbenes/isolation & purification/*metabolism ; },
abstract = {Efficient biosynthesis of the plant polyphenol pinosylvin, which has numerous applications in nutraceuticals and pharmaceuticals, is necessary to make biological production economically viable. To this end, an efficient Escherichia coli platform for pinosylvin production was developed via a rational modular design approach. Initially, different candidate pathway enzymes were screened to construct de novo pinosylvin pathway directly from D-glucose. A comparative analysis of pathway intermediate pools identified that this initial construct led to the intermediate cinnamic acid accumulation. The pinosylvin synthetic pathway was then divided into two new modules separated at cinnamic acid. Combinatorial optimization of transcriptional and translational levels of these two modules resulted in a 16-fold increase in pinosylvin titer. To further improve the concentration of the limiting precursor malonyl-CoA, the malonyl-CoA synthesis module based on clustered regularly interspaced short palindromic repeats interference was assembled and optimized with other two modules. The final pinosylvin titer was improved to 281 mg/L, which was the highest pinosylvin titer even directly from D-glucose without any additional precursor supplementation. The rational modular design approach described here could bolster our capabilities in synthetic biology for value-added chemical production.},
}

Base Sequence
CRISPR-Cas Systems
Cinnamates/metabolism
Escherichia coli/*enzymology/genetics
Escherichia coli Proteins/genetics/*metabolism
Gene Editing/methods
Glucose/metabolism
Malonyl Coenzyme A/metabolism
Metabolic Engineering/*methods
Metabolic Networks and Pathways/*genetics
Pinus sylvestris/chemistry
Plasmids/chemistry/metabolism
Polyphenols/*biosynthesis/isolation & purification
Stilbenes/isolation & purification/*metabolism

@article {pmid28374838,
year = {2017},
author = {Basu, S and Adams, L and Guhathakurta, S and Kim, YS},
title = {A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3'end using CRISPR-Cas9 genome editing technique.},
journal = {Scientific reports},
volume = {8},
number = {},
pages = {45883},
pmid = {28374838},
issn = {2045-2322},
support = {R21 NS088923/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Drug Evaluation, Preclinical ; Gene Editing/*methods ; Gene Expression Regulation ; Humans ; Luciferases/chemistry/genetics ; Parkinson Disease/*drug therapy/genetics/pathology ; Promoter Regions, Genetic ; *Transcription, Genetic ; Transfection ; alpha-Synuclein/*genetics ; },
abstract = {α-synuclein (α-SYN) is a major pathologic contributor to Parkinson's disease (PD). Multiplication of α-SYN encoding gene (SNCA) is correlated with early onset of the disease underlining the significance of its transcriptional regulation. Thus, monitoring endogenous transcription of SNCA is of utmost importance to understand PD pathology. We developed a stable cell line expressing α-SYN endogenously tagged with NanoLuc luciferase reporter using CRISPR/Cas9-mediated genome editing. This allows efficient measurement of transcriptional activity of α-SYN in its native epigenetic landscape which is not achievable using exogenous transfection-based luciferase reporter assays. The NanoLuc activity faithfully monitored the transcriptional regulation of SNCA following treatment with different drugs known to regulate α-SYN expression; while exogenous promoter-reporter assays failed to reproduce the similar outcomes. To our knowledge, this is the first report showing endogenous monitoring of α-SYN transcription, thus making it an efficient drug screening tool that can be used for therapeutic intervention in PD.},
}

CRISPR-Cas Systems/genetics
Cell Line
Drug Evaluation, Preclinical
Gene Editing/*methods
Gene Expression Regulation
Humans
Luciferases/chemistry/genetics
Parkinson Disease/*drug therapy/genetics/pathology
Promoter Regions, Genetic
*Transcription, Genetic
Transfection
alpha-Synuclein/*genetics