@article {pmid30319822,
year = {2018},
author = {Lone, BA and Karna, SKL and Ahmad, F and Shahi, N and Pokharel, YR},
title = {CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering.},
journal = {Genetics research international},
volume = {2018},
number = {},
pages = {3797214},
doi = {10.1155/2018/3797214},
pmid = {30319822},
issn = {2090-3154},
abstract = {Microbes use diverse defence strategies that allow them to withstand exposure to a variety of genome invaders such as bacteriophages and plasmids. One such defence strategy is the use of RNA guided endonuclease called CRISPR-associated (Cas) 9 protein. The Cas9 protein, derived from type II CRISPR/Cas system, has been adapted as a versatile tool for genome targeting and engineering due to its simplicity and high efficiency over the earlier tools such as ZFNs and TALENs. With recent advancements, CRISPR/Cas9 technology has emerged as a revolutionary tool for modulating the genome in living cells and inspires innovative translational applications in different fields. In this paper we review the developments and its potential uses in the CRISPR/Cas9 technology as well as recent advancements in genome engineering using CRISPR/Cas9.},
}

@article {pmid30269229,
year = {2018},
author = {Hong, KQ and Liu, DY and Chen, T and Wang, ZW},
title = {Recent advances in CRISPR/Cas9 mediated genome editing in Bacillus subtilis.},
journal = {World journal of microbiology & biotechnology},
volume = {34},
number = {10},
pages = {153},
doi = {10.1007/s11274-018-2537-1},
pmid = {30269229},
issn = {1573-0972},
support = {21576200//National Natural Science Foundation of China/ ; 21776209//National Natural Science Foundation of China/ ; 21621004//National Natural Science Foundation of China/ ; },
mesh = {Bacillus subtilis/*genetics ; Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases ; Gene Editing/*methods ; Genes, Bacterial/genetics ; Molecular Biology/methods ; },
abstract = {Genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used in various host cells. Its widespread adoption has been largely developed by the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR) system, which uses an easily customizable specificity RNA-guided DNA endonuclease, such as Cas9. Recently, CRISPR/Cas9 mediated genome engineering has been widely applied to model organisms, including Bacillus subtilis, enabling facile, rapid high-fidelity modification of endogenous native genes. Here, we reviewed the recent progress in B. subtilis gene editing using CRISPR/Cas9 based tools, and highlighted state-of-the-art strategies for design of CRISPR/Cas9 system. Finally, future perspectives on the use of CRISPR/Cas9 genome engineering for sequence-specific genome editing in B. subtilis are provided.},
}

Bacillus subtilis/*genetics
Bacterial Proteins/genetics
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Endonucleases
Gene Editing/*methods
Genes, Bacterial/genetics
Molecular Biology/methods

@article {pmid30180552,
year = {2018},
author = {Tian, H and Luo, J and Zhang, Z and Wu, J and Zhang, T and Busato, S and Huang, L and Song, N and Bionaz, M},
title = {CRISPR/Cas9-mediated Stearoyl-CoA Desaturase 1 (SCD1) Deficiency Affects Fatty Acid Metabolism in Goat Mammary Epithelial Cells.},
journal = {Journal of agricultural and food chemistry},
volume = {66},
number = {38},
pages = {10041-10052},
doi = {10.1021/acs.jafc.8b03545},
pmid = {30180552},
issn = {1520-5118},
mesh = {Animals ; Animals, Genetically Modified/*genetics/metabolism ; CRISPR-Cas Systems ; Epithelial Cells/*enzymology/metabolism ; Fatty Acids/*metabolism ; Female ; Gene Deletion ; Gene Knockout Techniques/*methods ; Goats/*genetics/metabolism ; Lipogenesis ; Mammary Glands, Animal/cytology/*enzymology/metabolism ; Milk/chemistry/metabolism ; Stearoyl-CoA Desaturase/*deficiency/genetics ; Sterol Regulatory Element Binding Protein 1/genetics/metabolism ; Triglycerides/metabolism ; },
abstract = {Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Δ9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC. We successfully introduced nucleotide deletions and mutations in the SCD1 gene locus through the NHEJ pathway and disrupted its second exon via insertion of an EGFP-PuroR segment using the HDR pathway. In clones derived from the latter, gene- and protein-expression data indicated that we obtained a monoallelic SCD1 knockout. A T7EN1-mediated assay revealed no off-targets in the surveyed sites. The contents of triacylglycerol and cholesterol and the desaturase index were significantly decreased as a consequence of SCD1 knockout. The deletion of SCD1 decreased the expression of other genes involved in de novo fatty acid synthesis, including SREBF1 and FASN, as well the fatty acid transporters FABP3 and FABP4. The downregulation of these genes partly explains the decrease of intracellular triacylglycerols. Our results indicate a successful SCD1 knockout in goat mammary cells using CRISPR-Cas9. The demonstration of the successful use of CRISPR-Cas9 in GMEC is an important step to producing transgenic goats to study mammary biology in vivo.},
}

Animals
Animals, Genetically Modified/*genetics/metabolism
CRISPR-Cas Systems
Epithelial Cells/*enzymology/metabolism
Fatty Acids/*metabolism
Female
Gene Deletion
Gene Knockout Techniques/*methods
Goats/*genetics/metabolism
Lipogenesis
Mammary Glands, Animal/cytology/*enzymology/metabolism
Milk/chemistry/metabolism
Stearoyl-CoA Desaturase/*deficiency/genetics
Sterol Regulatory Element Binding Protein 1/genetics/metabolism
Triglycerides/metabolism

@article {pmid30082270,
year = {2018},
author = {Jardin, N and Giudicelli, F and Ten Martín, D and Vitrac, A and De Gois, S and Allison, R and Houart, C and Reid, E and Hazan, J and Fassier, C},
title = {BMP- and neuropilin 1-mediated motor axon navigation relies on spastin alternative translation.},
journal = {Development (Cambridge, England)},
volume = {145},
number = {17},
pages = {},
doi = {10.1242/dev.162701},
pmid = {30082270},
issn = {1477-9129},
support = {MR/M00046X/1//Medical Research Council/United Kingdom ; },
mesh = {Animals ; Axons/metabolism ; Bone Morphogenetic Proteins/*metabolism ; COS Cells ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Movement/genetics ; Cercopithecus aethiops ; GTP-Binding Proteins/metabolism ; Gene Knockout Techniques ; Humans ; Membrane Proteins/metabolism ; Motor Neurons/*cytology ; Neuropilin-1/*metabolism ; Protein Isoforms/genetics ; Spastic Paraplegia, Hereditary/genetics ; Spastin/biosynthesis/*genetics ; Zebrafish/*embryology ; Zebrafish Proteins/biosynthesis/*genetics ; },
abstract = {Functional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4-encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motor neuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 spastin mutant. We next showed that M1 spastin, together with HSP proteins atlastin 1 and NIPA1, drives motor axon targeting by repressing BMP signalling, whereas M87 spastin acts downstream of neuropilin 1 to control motor neuron migration. Our data therefore suggest that defective BMP and neuropilin 1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.},
}

Animals
Axons/metabolism
Bone Morphogenetic Proteins/*metabolism
COS Cells
CRISPR-Cas Systems/genetics
Cell Line
Cell Movement/genetics
Cercopithecus aethiops
GTP-Binding Proteins/metabolism
Gene Knockout Techniques
Humans
Membrane Proteins/metabolism
Motor Neurons/*cytology
Neuropilin-1/*metabolism
Protein Isoforms/genetics
Spastic Paraplegia, Hereditary/genetics
Spastin/biosynthesis/*genetics
Zebrafish/*embryology
Zebrafish Proteins/biosynthesis/*genetics

@article {pmid29489336,
year = {2018},
author = {Nguyen, NT and He, L and Martinez-Moczygemba, M and Huang, Y and Zhou, Y},
title = {Rewiring Calcium Signaling for Precise Transcriptional Reprogramming.},
journal = {ACS synthetic biology},
volume = {7},
number = {3},
pages = {814-821},
doi = {10.1021/acssynbio.7b00467},
pmid = {29489336},
issn = {2161-5063},
support = {R01 GM112003/GM/NIGMS NIH HHS/United States ; R21 GM126532/GM/NIGMS NIH HHS/United States ; R01 HL134780/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Calcium Release Activated Calcium Channels/metabolism ; Calcium Signaling/*genetics ; Cell Nucleus/metabolism ; Gene Expression Regulation/radiation effects ; HEK293 Cells ; HeLa Cells ; Humans ; Light ; Optogenetics ; Protein Transport ; Synthetic Biology/*methods ; *Transcription, Genetic ; },
abstract = {Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca2+-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.},
}

CRISPR-Cas Systems/genetics
Calcium/metabolism
Calcium Release Activated Calcium Channels/metabolism
Calcium Signaling/*genetics
Cell Nucleus/metabolism
Gene Expression Regulation/radiation effects
HEK293 Cells
HeLa Cells
Humans
Light
Optogenetics
Protein Transport
Synthetic Biology/*methods
*Transcription, Genetic

@article {pmid29486117,
year = {2018},
author = {Qiu, XY and Zhu, LY and Zhu, CS and Ma, JX and Hou, T and Wu, XM and Xie, SS and Min, L and Tan, DA and Zhang, DY and Zhu, L},
title = {Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9.},
journal = {ACS synthetic biology},
volume = {7},
number = {3},
pages = {807-813},
doi = {10.1021/acssynbio.7b00446},
pmid = {29486117},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Carcinoma, Non-Small-Cell Lung/blood/genetics ; *Costs and Cost Analysis ; Genetic Techniques/*economics ; Horseradish Peroxidase/metabolism ; Humans ; Lung Neoplasms/blood/genetics ; MicroRNAs/*analysis/*economics/genetics ; RNA Probes/metabolism ; },
abstract = {MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases.},
}

CRISPR-Cas Systems/*genetics
Carcinoma, Non-Small-Cell Lung/blood/genetics
*Costs and Cost Analysis
Genetic Techniques/*economics
Horseradish Peroxidase/metabolism
Humans
Lung Neoplasms/blood/genetics
MicroRNAs/*analysis/*economics/genetics
RNA Probes/metabolism

@article {pmid29467179,
year = {2018},
author = {Rauscher, B and Heigwer, F and Henkel, L and Hielscher, T and Voloshanenko, O and Boutros, M},
title = {Toward an integrated map of genetic interactions in cancer cells.},
journal = {Molecular systems biology},
volume = {14},
number = {2},
pages = {e7656},
pmid = {29467179},
issn = {1744-4292},
mesh = {CRISPR-Cas Systems ; Epistasis, Genetic ; *Gene Regulatory Networks ; Genetic Variation ; Humans ; Neoplasms/*genetics ; Systems Biology/*methods ; Wnt Signaling Pathway ; },
abstract = {Cancer genomes often harbor hundreds of molecular aberrations. Such genetic variants can be drivers or passengers of tumorigenesis and create vulnerabilities for potential therapeutic exploitation. To identify genotype-dependent vulnerabilities, forward genetic screens in different genetic backgrounds have been conducted. We devised MINGLE, a computational framework to integrate CRISPR/Cas9 screens originating from different libraries building on approaches pioneered for genetic network discovery in model organisms. We applied this method to integrate and analyze data from 85 CRISPR/Cas9 screens in human cancer cells combining functional data with information on genetic variants to explore more than 2.1 million gene-background relationships. In addition to known dependencies, we identified new genotype-specific vulnerabilities of cancer cells. Experimental validation of predicted vulnerabilities identified GANAB and PRKCSH as new positive regulators of Wnt/β-catenin signaling. By clustering genes with similar genetic interaction profiles, we drew the largest genetic network in cancer cells to date. Our scalable approach highlights how diverse genetic screens can be integrated to systematically build informative maps of genetic interactions in cancer, which can grow dynamically as more data are included.},
}

CRISPR-Cas Systems
Epistasis, Genetic
*Gene Regulatory Networks
Genetic Variation
Humans
Neoplasms/*genetics
Systems Biology/*methods
Wnt Signaling Pathway

@article {pmid28945226,
year = {2018},
author = {Bian, X and Gao, J and Luo, F and Rui, C and Zheng, T and Wang, D and Wang, Y and Roberts, TM and Liu, P and Zhao, JJ and Cheng, H},
title = {PTEN deficiency sensitizes endometrioid endometrial cancer to compound PARP-PI3K inhibition but not PARP inhibition as monotherapy.},
journal = {Oncogene},
volume = {37},
number = {3},
pages = {341-351},
doi = {10.1038/onc.2017.326},
pmid = {28945226},
issn = {1476-5594},
support = {R01 CA187918/CA/NCI NIH HHS/United States ; R01 CA172461/CA/NCI NIH HHS/United States ; P50 CA168504/CA/NCI NIH HHS/United States ; R35 CA210057/CA/NCI NIH HHS/United States ; P50 CA165962/CA/NCI NIH HHS/United States ; },
mesh = {Aminopyridines/pharmacology/therapeutic use ; Animals ; Antineoplastic Combined Chemotherapy Protocols/*pharmacology/therapeutic use ; CRISPR-Cas Systems ; Carcinoma, Endometrioid/*drug therapy/genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Drug Resistance, Neoplasm ; Endometrial Neoplasms/*drug therapy/genetics/pathology ; Endometrium/drug effects/pathology ; Female ; Humans ; Mice ; Mice, Transgenic ; Morpholines/pharmacology/therapeutic use ; Neoplasms, Experimental/drug therapy/genetics/pathology ; PTEN Phosphohydrolase/*deficiency/genetics ; Phosphatidylinositol 3-Kinases/antagonists & inhibitors ; Phthalazines/pharmacology/therapeutic use ; Piperazines/pharmacology/therapeutic use ; Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology/therapeutic use ; Protein Kinase Inhibitors/*pharmacology/therapeutic use ; Recombinational DNA Repair/drug effects ; Spheroids, Cellular/drug effects/pathology ; },
abstract = {Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising cancer therapeutics especially for tumors with deficient homologous recombination (HR) repair. However, as HR-deficient tumors represent only a small fraction of endometrial cancers, the therapeutic utility of PARP inhibitors is limited in this disease. Somatic loss of phosphatase and tensin homolog (PTEN), a tumor suppressor that counteracts phosphoinositide 3-kinase (PI3K) activity, is one of the most common genetic aberrations in endometrioid endometrial cancer. While previous works have identified the role of PTEN in DNA double-strand break repair, vulnerabilities of PTEN-deficient endometrioid endometrial cancers to PARP inhibition remain controversial. Here we find that PTEN-deficient endometrioid endometrial cancer cells are not responsive to PARP inhibitor Olaparib alone, but instead show superior sensitivity to compound inhibition with PI3K inhibitor BKM120, as evidenced by reduced clonogenic cell growth and three-dimensional (3D) spheroid disintegration. Mechanistically, PI3K blockade by BKM120 attenuated HR competency with γH2AX accumulation and reduced RAD51 and BRCA1 expression in Ishikawa, AN3CA and Nou-1 cells, but the same combination treatment led to enhanced phosphorylation of DNA-PK, a non-homologous end joining repair protein, in Hec-108 cells. Furthermore, we show that CRISPR/Cas9-mediated PTEN depletion rendered PTEN wild-type Hec-1A endometrioid endometrial cancer cells responsive to combined inhibition of PARP/PI3K, with concomitantly induced DNA damage accumulation and repair defects. The combination of BKM120 and Olaparib cooperated to inhibit tumor growth in a genetic mouse model of Pten-deficient endometrioid endometrial cancer. Together, these results suggest PI3K inhibition may be a plausible approach to expand the utility of PARP inhibitors to endometrioid endometrial cancers in a PTEN-deficient setting.},
}

Aminopyridines/pharmacology/therapeutic use
Animals
Antineoplastic Combined Chemotherapy Protocols/*pharmacology/therapeutic use
CRISPR-Cas Systems
Carcinoma, Endometrioid/*drug therapy/genetics/pathology
Cell Line, Tumor
Cell Proliferation/drug effects
Drug Resistance, Neoplasm
Endometrial Neoplasms/*drug therapy/genetics/pathology
Endometrium/drug effects/pathology
Female
Humans
Mice
Mice, Transgenic
Morpholines/pharmacology/therapeutic use
Neoplasms, Experimental/drug therapy/genetics/pathology
PTEN Phosphohydrolase/*deficiency/genetics
Phosphatidylinositol 3-Kinases/antagonists & inhibitors
Phthalazines/pharmacology/therapeutic use
Piperazines/pharmacology/therapeutic use
Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors
Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology/therapeutic use
Protein Kinase Inhibitors/*pharmacology/therapeutic use
Recombinational DNA Repair/drug effects
Spheroids, Cellular/drug effects/pathology

@article {pmid28071690,
year = {2017},
author = {Park, KE and Kaucher, AV and Powell, A and Waqas, MS and Sandmaier, SE and Oatley, MJ and Park, CH and Tibary, A and Donovan, DM and Blomberg, LA and Lillico, SG and Whitelaw, CB and Mileham, A and Telugu, BP and Oatley, JM},
title = {Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {40176},
doi = {10.1038/srep40176},
pmid = {28071690},
issn = {2045-2322},
support = {P51 OD011092/OD/NIH HHS/United States ; },
mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems ; Fertility ; *Gene Knockout Techniques ; Infertility, Male ; Male ; RNA-Binding Proteins/*genetics ; Sus scrofa/*genetics ; },
abstract = {Genome editing tools have revolutionized the generation of genetically modified animals including livestock. In particular, the domestic pig is a proven model of human physiology and an agriculturally important species. In this study, we utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. We found that NANOS2 knockout pigs phenocopy knockout mice with male specific germline ablation but other aspects of testicular development are normal. Moreover, male pigs with one intact NANOS2 allele and female knockout pigs are fertile. From an agriculture perspective, NANOS2 knockout male pigs are expected to serve as an ideal surrogate for transplantation of donor spermatogonial stem cells to expand the availability of gametes from genetically desirable sires.},
}

Animals
*Animals, Genetically Modified
CRISPR-Cas Systems
Fertility
*Gene Knockout Techniques
Infertility, Male
Male
RNA-Binding Proteins/*genetics
Sus scrofa/*genetics

@article {pmid28053351,
year = {2017},
author = {Khan, SA and Reichelt, M and Heckel, DG},
title = {Functional analysis of the ABCs of eye color in Helicoverpa armigera with CRISPR/Cas9-induced mutations.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {40025},
doi = {10.1038/srep40025},
pmid = {28053351},
issn = {2045-2322},
mesh = {ATP-Binding Cassette Transporters/*genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; DNA Mutational Analysis ; Eye Color/*genetics ; Gene Knockout Techniques ; Lepidoptera/*genetics ; Pigments, Biological/*metabolism ; },
abstract = {Many insect pigments are localized in subcellular pigment granules, and transport of pigment precursors from the cytoplasm is accomplished by ABC proteins. Drosophila melanogaster has three half-transporter genes (white, scarlet, and brown, all affecting eye pigments) and Bombyx mori has a fourth (ok). The White, Brown, Scarlet and Ok proteins each have one transmembrane and one cytoplasmic domain and they heterodimerize to form functional transporters with different substrate specificities. We used CRISPR/Cas9 to create somatic and germ-line knockout mutations of these four genes in the noctuid moth Helicoverpa armigera. Somatic knockouts of white block pigmentation of the egg, first instar larva and adult eye, but germ-line knockouts of white are recessive lethal in the embryo. Knockouts of scarlet are viable and produce pigmentless first instar larvae and yellow adult eyes lacking xanthommatin. Knockouts of brown show no phenotypic effects on viability or pigmentation. Knockouts of ok are viable and produce translucent larval cuticle and black eyes. CRISPR/Cas9-induced mutations are a useful tool for analyzing how essential and non-essential genes interact to produce the diversity of insect pigmentation patterns found in nature.},
}

ATP-Binding Cassette Transporters/*genetics/*metabolism
Animals
CRISPR-Cas Systems
DNA Mutational Analysis
Eye Color/*genetics
Gene Knockout Techniques
Lepidoptera/*genetics
Pigments, Biological/*metabolism

@article {pmid30317145,
year = {2018},
author = {Bron, PA and Marcelli, B and Mulder, J and van der Els, S and Morawska, LP and Kuipers, OP and Kok, J and Kleerebezem, M},
title = {Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid bacteria.},
journal = {Current opinion in biotechnology},
volume = {56},
number = {},
pages = {61-68},
doi = {10.1016/j.copbio.2018.09.004},
pmid = {30317145},
issn = {1879-0429},
abstract = {The ever-expanding genomic insight in natural diversity of lactic acid bacteria (LAB) has revived the industrial interest in traditional and natural genetic mobilization methodologies. Here, we review recent advances in horizontal gene transfer processes in LAB, including natural competence, conjugation, and phage transduction. In addition, we envision the possibilities for industrial strain improvement arising from the recent discoveries of molecular exchanges between bacteria through nanotubes and extracellular vesicles, as well as the constantly expanding genome editing possibilities using the CRISPR-Cas technology.},
}

@article {pmid30201719,
year = {2018},
author = {Viegas, J},
title = {Profile of Dana Carroll.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {38},
pages = {9331-9333},
doi = {10.1073/pnas.1813829115},
pmid = {30201719},
issn = {1091-6490},
mesh = {CRISPR-Cas Systems ; Gene Editing/*history/*methods ; History, 20th Century ; History, 21st Century ; Homologous Recombination ; Transcription Activator-Like Effector Nucleases/metabolism ; United States ; Zinc Finger Nucleases/metabolism ; },
}

CRISPR-Cas Systems
Gene Editing/*history/*methods
History, 20th Century
History, 21st Century
Homologous Recombination
Transcription Activator-Like Effector Nucleases/metabolism
United States
Zinc Finger Nucleases/metabolism

@article {pmid30185555,
year = {2018},
author = {Wall, RJ and Rico, E and Lukac, I and Zuccotto, F and Elg, S and Gilbert, IH and Freund, Y and Alley, MRK and Field, MC and Wyllie, S and Horn, D},
title = {Clinical and veterinary trypanocidal benzoxaboroles target CPSF3.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {38},
pages = {9616-9621},
doi = {10.1073/pnas.1807915115},
pmid = {30185555},
issn = {1091-6490},
support = {105021/Z/14/Z//Wellcome Trust/United Kingdom ; MR/K008749/1//Medical Research Council/United Kingdom ; //Wellcome Trust/United Kingdom ; 100320/Z/12/Z//Wellcome Trust/United Kingdom ; 203134/Z/16/Z//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Benzamides/pharmacology/therapeutic use ; Boron Compounds/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Cell Nucleus/genetics/metabolism ; Cleavage And Polyadenylation Specificity Factor/*antagonists & inhibitors/genetics/metabolism ; Drug Resistance/drug effects/genetics ; Gene Knockdown Techniques ; Gene Library ; High-Throughput Screening Assays/methods ; Humans ; Molecular Docking Simulation ; Protozoan Proteins/*antagonists & inhibitors/genetics/metabolism ; RNA Processing, Post-Transcriptional/drug effects ; RNA, Messenger/metabolism ; RNA, Protozoan/metabolism ; Trypanocidal Agents/*pharmacology/therapeutic use ; Trypanosoma brucei brucei/drug effects/*physiology ; Trypanosomiasis, African/*prevention & control/transmission/veterinary ; Valine/analogs & derivatives/pharmacology/therapeutic use ; },
abstract = {African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.},
}

Animals
Benzamides/pharmacology/therapeutic use
Boron Compounds/pharmacology/therapeutic use
CRISPR-Cas Systems
Cell Nucleus/genetics/metabolism
Cleavage And Polyadenylation Specificity Factor/*antagonists & inhibitors/genetics/metabolism
Drug Resistance/drug effects/genetics
Gene Knockdown Techniques
Gene Library
High-Throughput Screening Assays/methods
Humans
Molecular Docking Simulation
Protozoan Proteins/*antagonists & inhibitors/genetics/metabolism
RNA Processing, Post-Transcriptional/drug effects
RNA, Messenger/metabolism
RNA, Protozoan/metabolism
Trypanocidal Agents/*pharmacology/therapeutic use
Trypanosoma brucei brucei/drug effects/*physiology
Trypanosomiasis, African/*prevention & control/transmission/veterinary
Valine/analogs & derivatives/pharmacology/therapeutic use

@article {pmid29980799,
year = {2018},
author = {Ungewiß, H and Rötzer, V and Meir, M and Fey, C and Diefenbacher, M and Schlegel, N and Waschke, J},
title = {Dsg2 via Src-mediated transactivation shapes EGFR signaling towards cell adhesion.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {75},
number = {22},
pages = {4251-4268},
doi = {10.1007/s00018-018-2869-x},
pmid = {29980799},
issn = {1420-9071},
support = {SPP1782//Deutsche Forschungsgemeinschaft/ ; SPP1782//Deutsche Forschungsgemeinschaft/ ; },
mesh = {CRISPR-Cas Systems/genetics ; Caco-2 Cells ; Cell Adhesion ; Cell Line, Tumor ; Cell Proliferation ; Desmoglein 2/deficiency/genetics/*metabolism ; Desmosomes/metabolism ; Humans ; Intestinal Mucosa/cytology/metabolism ; Microscopy, Atomic Force ; Protein Binding ; Receptor, Epidermal Growth Factor/*metabolism ; Signal Transduction ; Transcriptional Activation ; p38 Mitogen-Activated Protein Kinases/metabolism ; src-Family Kinases/genetics/*metabolism ; },
abstract = {Rapidly renewing epithelial tissues such as the intestinal epithelium require precise tuning of intercellular adhesion and proliferation to preserve barrier integrity. Here, we provide evidence that desmoglein 2 (Dsg2), an adhesion molecule of desmosomes, controls cell adhesion and proliferation via epidermal growth factor receptor (EGFR) signaling. Dsg2 is required for EGFR localization at intercellular junctions as well as for Src-mediated EGFR activation. Src binds to EGFR and is required for localization of EGFR and Dsg2 to cell-cell contacts. EGFR is critical for cell adhesion and barrier recovery. In line with this, Dsg2-deficient enterocytes display impaired barrier properties and increased cell proliferation. Mechanistically, Dsg2 directly interacts with EGFR and undergoes heterotypic-binding events on the surface of living enterocytes via its extracellular domain as revealed by atomic force microscopy. Thus, our study reveals a new mechanism by which Dsg2 via Src shapes EGFR function towards cell adhesion.},
}

CRISPR-Cas Systems/genetics
Caco-2 Cells
Cell Adhesion
Cell Line, Tumor
Cell Proliferation
Desmoglein 2/deficiency/genetics/*metabolism
Desmosomes/metabolism
Humans
Intestinal Mucosa/cytology/metabolism
Microscopy, Atomic Force
Protein Binding
Receptor, Epidermal Growth Factor/*metabolism
Signal Transduction
Transcriptional Activation
p38 Mitogen-Activated Protein Kinases/metabolism
src-Family Kinases/genetics/*metabolism

@article {pmid29233915,
year = {2018},
author = {Li, M and Akbari, OS and White, BJ},
title = {Highly Efficient Site-Specific Mutagenesis in Malaria Mosquitoes Using CRISPR.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {2},
pages = {653-658},
doi = {10.1534/g3.117.1134},
pmid = {29233915},
issn = {2160-1836},
support = {K22 AI113060/AI/NIAID NIH HHS/United States ; R01 AI113248/AI/NIAID NIH HHS/United States ; R21 AI115271/AI/NIAID NIH HHS/United States ; R21 AI123937/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Anopheles/classification/*genetics/physiology ; *CRISPR-Cas Systems ; Humans ; Malaria/parasitology ; Mosquito Vectors/classification/*genetics/physiology ; Mutagenesis, Site-Directed/*methods ; Species Specificity ; },
abstract = {Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus, A. coluzzii, and A. funestus When guide RNAs (gRNAs) and Cas9 protein are injected at high concentration, germline mutations are common and usually biallelic, allowing for the rapid creation of stable mutant lines for reverse genetic analysis. Our protocol should enable researchers to dissect the molecular and cellular basis of anopheline traits critical to successful disease transmission, potentially exposing new targets for malaria control.},
}

Animals
Anopheles/classification/*genetics/physiology
*CRISPR-Cas Systems
Humans
Malaria/parasitology
Mosquito Vectors/classification/*genetics/physiology
Mutagenesis, Site-Directed/*methods
Species Specificity

@article {pmid28560349,
year = {2017},
author = {Miyamoto, T and Lo, PHY and Saichi, N and Ueda, K and Hirata, M and Tanikawa, C and Matsuda, K},
title = {Argininosuccinate synthase 1 is an intrinsic Akt repressor transactivated by p53.},
journal = {Science advances},
volume = {3},
number = {5},
pages = {e1603204},
doi = {10.1126/sciadv.1603204},
pmid = {28560349},
issn = {2375-2548},
mesh = {Animals ; Apoptosis/genetics/radiation effects ; Argininosuccinate Synthase/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/genetics/radiation effects ; Humans ; Intestine, Small/metabolism/pathology ; Mice ; Mice, Knockout ; Proto-Oncogene Proteins c-akt/genetics/*metabolism ; Signal Transduction/genetics/*radiation effects ; Transcriptional Activation/genetics/*radiation effects ; Tumor Suppressor Protein p53/genetics/*metabolism ; X-Rays/*adverse effects ; },
abstract = {The transcription factor p53 is at the core of a built-in tumor suppression system that responds to varying degrees of stress input and is deregulated in most human cancers. Befitting its role in maintaining cellular fitness and fidelity, p53 regulates an appropriate set of target genes in response to cellular stresses. However, a comprehensive understanding of this scheme has not been accomplished. We show that argininosuccinate synthase 1 (ASS1), a citrulline-aspartate ligase in de novo arginine synthesis pathway, was directly transactivated by p53 in response to genotoxic stress, resulting in the rearrangement of arginine metabolism. Furthermore, we found that x-ray irradiation promoted the systemic induction of Ass1 and concomitantly increased plasma arginine levels in p53+/+ mice but not in p53-/- mice. Notably, Ass1+/- mice exhibited hypersensitivity to whole-body irradiation owing to increased apoptosis in the small intestinal crypts. Analyses of ASS1-deficient cells generated using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) system revealed that ASS1 plays a pivotal role in limiting Akt phosphorylation. In addition, aberrant activation of Akt resulting from ASS1 loss disrupted Akt-mediated cell survival signaling activity under genotoxic stress. Building on these results, we demonstrated that p53 induced an intrinsic Akt repressor, ASS1, and the perturbation of ASS1 expression rendered cells susceptible to genotoxic stress. Our findings uncover a new function of p53 in the regulation of Akt signaling and reveal how p53, ASS1, and Akt are interrelated to each other.},
}

Animals
Apoptosis/genetics/radiation effects
Argininosuccinate Synthase/genetics/*metabolism
CRISPR-Cas Systems
Cell Line, Tumor
Cell Survival/genetics/radiation effects
Humans
Intestine, Small/metabolism/pathology
Mice
Mice, Knockout
Proto-Oncogene Proteins c-akt/genetics/*metabolism
Signal Transduction/genetics/*radiation effects
Transcriptional Activation/genetics/*radiation effects
Tumor Suppressor Protein p53/genetics/*metabolism
X-Rays/*adverse effects

@article {pmid28560324,
year = {2017},
author = {Drury, DW and Dapper, AL and Siniard, DJ and Zentner, GE and Wade, MJ},
title = {CRISPR/Cas9 gene drives in genetically variable and nonrandomly mating wild populations.},
journal = {Science advances},
volume = {3},
number = {5},
pages = {e1601910},
doi = {10.1126/sciadv.1601910},
pmid = {28560324},
issn = {2375-2548},
support = {R01 GM084238/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Genetic Variation ; *Models, Genetic ; Reproduction/genetics ; Tribolium/*genetics ; },
abstract = {Synthetic gene drives based on CRISPR/Cas9 have the potential to control, alter, or suppress populations of crop pests and disease vectors, but it is unclear how they will function in wild populations. Using genetic data from four populations of the flour beetle Tribolium castaneum, we show that most populations harbor genetic variants in Cas9 target sites, some of which would render them immune to drive (ITD). We show that even a rare ITD allele can reduce or eliminate the efficacy of a CRISPR/Cas9-based synthetic gene drive. This effect is equivalent to and accentuated by mild inbreeding, which is a characteristic of many disease-vectoring arthropods. We conclude that designing such drives will require characterization of genetic variability and the mating system within and among targeted populations.},
}

Animals
*CRISPR-Cas Systems
*Genetic Variation
*Models, Genetic
Reproduction/genetics
Tribolium/*genetics

@article {pmid28508063,
year = {2017},
author = {Lee, B and Sahoo, A and Marchica, J and Holzhauser, E and Chen, X and Li, JL and Seki, T and Govindarajan, SS and Markey, FB and Batish, M and Lokhande, SJ and Zhang, S and Ray, A and Perera, RJ},
title = {The long noncoding RNA SPRIGHTLY acts as an intranuclear organizing hub for pre-mRNA molecules.},
journal = {Science advances},
volume = {3},
number = {5},
pages = {e1602505},
doi = {10.1126/sciadv.1602505},
pmid = {28508063},
issn = {2375-2548},
support = {DP5 OD012160/OD/NIH HHS/United States ; P30 CA030199/CA/NCI NIH HHS/United States ; R03 CA165184/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Melanoma/genetics ; Mice ; Mice, SCID ; RNA Precursors/*genetics ; RNA, Long Noncoding/*genetics ; },
abstract = {Molecular mechanisms by which long noncoding RNA (lncRNA) molecules may influence cancerous condition are poorly understood. The aberrant expression of SPRIGHTLY lncRNA, encoded within the drosophila gene homolog Sprouty-4 intron, is correlated with a variety of cancers, including human melanomas. We demonstrate by SHAPE-seq and dChIRP that SPRIGHTLY RNA secondary structure has a core pseudoknotted domain. This lncRNA interacts with the intronic regions of six pre-mRNAs: SOX5, SMYD3, SND1, MEOX2, DCTN6, and RASAL2, all of which have cancer-related functions. Hemizygous knockout of SPRIGHTLY by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 in melanoma cells significantly decreases SPRIGHTLY lncRNA levels, simultaneously decreases the levels of its interacting pre-mRNA molecules, and decreases anchorage-independent growth rate of cells and the rate of in vivo tumor growth in mouse xenografts. These results provide the first demonstration of an lncRNA's three-dimensional coordinating role in facilitating cancer-related gene expression in human melanomas.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
Melanoma/genetics
Mice
Mice, SCID
RNA Precursors/*genetics
RNA, Long Noncoding/*genetics

@article {pmid28045112,
year = {2017},
author = {Ren, W and Wang, C and Wang, Q and Zhao, D and Zhao, K and Sun, D and Liu, X and Han, C and Hou, J and Li, X and Zhang, Q and Cao, X and Li, N},
title = {Bromodomain protein Brd3 promotes Ifnb1 transcription via enhancing IRF3/p300 complex formation and recruitment to Ifnb1 promoter in macrophages.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {39986},
doi = {10.1038/srep39986},
pmid = {28045112},
issn = {2045-2322},
mesh = {Acetylation ; Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; E1A-Associated p300 Protein/*metabolism ; Histones/metabolism ; Interferon Regulatory Factor-3/*metabolism ; Interferon-beta/genetics/*metabolism ; Macrophages, Peritoneal/cytology/metabolism/virology ; Mice ; Mice, Inbred C57BL ; Nuclear Proteins/antagonists & inhibitors/genetics/*metabolism ; Promoter Regions, Genetic ; RAW 264.7 Cells ; RNA Interference ; RNA, Small Interfering/metabolism ; Transcriptional Activation ; Viruses/pathogenicity ; },
abstract = {As members of bromodomain and extra-terminal motif protein family, bromodomain-containing proteins regulate a wide range of biological processes including protein scaffolding, mitosis, cell cycle progression and transcriptional regulation. The function of these bromodomain proteins (Brds) in innate immune response has been reported but the role of Brd3 remains unclear. Here we find that virus infection significantly downregulate Brd3 expression in macrophages and Brd3 knockout inhibits virus-triggered IFN-β production. Brd3 interacts with both IRF3 and p300, increases p300-mediated acetylation of IRF3, and enhances the association of IRF3 with p300 upon virus infection. Importantly, Brd3 promotes the recruitment of IRF3/p300 complex to the promoter of Ifnb1, and increases the acetylation of histone3/histone4 within the Ifnb1 promoter, leading to the enhancement of type I interferon production. Therefore, our work indicated that Brd3 may act as a coactivator in IRF3/p300 transcriptional activation of Ifnb1 and provided new epigenetic mechanistic insight into the efficient activation of the innate immune response.},
}

Acetylation
Animals
CRISPR-Cas Systems/genetics
Cells, Cultured
E1A-Associated p300 Protein/*metabolism
Histones/metabolism
Interferon Regulatory Factor-3/*metabolism
Interferon-beta/genetics/*metabolism
Macrophages, Peritoneal/cytology/metabolism/virology
Mice
Mice, Inbred C57BL
Nuclear Proteins/antagonists & inhibitors/genetics/*metabolism
Promoter Regions, Genetic
RAW 264.7 Cells
RNA Interference
RNA, Small Interfering/metabolism
Transcriptional Activation
Viruses/pathogenicity

@article {pmid30297904,
year = {2018},
author = {Villiger, L and Grisch-Chan, HM and Lindsay, H and Ringnalda, F and Pogliano, CB and Allegri, G and Fingerhut, R and Häberle, J and Matos, J and Robinson, MD and Thöny, B and Schwank, G},
title = {Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.},
journal = {Nature medicine},
volume = {24},
number = {10},
pages = {1519-1525},
doi = {10.1038/s41591-018-0209-1},
pmid = {30297904},
issn = {1546-170X},
abstract = {CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.},
}

@article {pmid30124643,
year = {2018},
author = {Xu, L and Gao, Y and Lau, YS and Han, R},
title = {Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {138},
pages = {},
doi = {10.3791/57560},
pmid = {30124643},
issn = {1940-087X},
support = {R01 HL116546/HL/NHLBI NIH HHS/United States ; R01 AR064241/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Dystrophin/*metabolism ; Gene Editing/*methods ; Humans ; Mice ; Muscular Dystrophy, Duchenne/*genetics/*metabolism ; },
abstract = {The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.},
}

Animals
CRISPR-Cas Systems/*genetics
Dystrophin/*metabolism
Gene Editing/*methods
Humans
Mice
Muscular Dystrophy, Duchenne/*genetics/*metabolism

@article {pmid29884745,
year = {2018},
author = {Germoglio, M and Adamo, A},
title = {A Role in Apoptosis Regulation for the rad-51 Gene of Caenorhabditis elegans.},
journal = {Genetics},
volume = {209},
number = {4},
pages = {1017-1028},
doi = {10.1534/genetics.118.301152},
pmid = {29884745},
issn = {1943-2631},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics/*metabolism ; Caenorhabditis elegans Proteins/*genetics/*metabolism ; DNA Damage ; Female ; Germ Cells/*cytology/metabolism ; Male ; Mutation ; Rad51 Recombinase/*genetics/*metabolism ; },
abstract = {The evolutionarily conserved RAD-51 protein is essential for homologous recombination in the germ line as well as homologous repair of DNA double-strand breaks in all eukaryotic cells. In the nematode Caenorhabditis elegans, the rad-51 gene is transcribed into messenger RNAs potentially coding three alternative protein isoforms. Null rad-51 alleles display embryonic lethality, severe defects in chromosome structure, and high levels of germ line apoptosis. To dissect its functions, we genetically modified the C. elegans rad-51 gene by clustered regularly interspaced short palindromic repeats/Cas9 genome-editing technology, obtaining a separation-of-function (sfi-) mutant allele that only disrupts the long-transcript isoform. This mutant shows no defects in an otherwise wild-type meiosis and is able to activate physiological germ cell death, which occurs at the late pachytene stage. However, although the mutant is competent in DNA damage checkpoint activation after exposure to ionizing radiation, it is defective for induction of DNA damage-induced apoptosis in meiotic germ cells. These results suggest that RAD-51 plays a novel role in germ line apoptosis independent of RAD-51-mediated strand invasion for homologous recombination.},
}

Animals
Apoptosis
CRISPR-Cas Systems
Caenorhabditis elegans/genetics/*metabolism
Caenorhabditis elegans Proteins/*genetics/*metabolism
DNA Damage
Female
Germ Cells/*cytology/metabolism
Male
Mutation
Rad51 Recombinase/*genetics/*metabolism

@article {pmid29729270,
year = {2018},
author = {Chen, G and Zhang, D and Zhang, L and Feng, G and Zhang, B and Wu, Y and Li, W and Zhang, Y and Hu, B},
title = {RBM14 is indispensable for pluripotency maintenance and mesoderm development of mouse embryonic stem cells.},
journal = {Biochemical and biophysical research communications},
volume = {501},
number = {1},
pages = {259-265},
doi = {10.1016/j.bbrc.2018.04.231},
pmid = {29729270},
issn = {1090-2104},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Gene Knockout Techniques ; Mesoderm/*embryology/*metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Mouse Embryonic Stem Cells/*cytology/*metabolism/transplantation ; Transcription Factors/deficiency/genetics/*metabolism ; Transcriptome ; Transforming Growth Factor beta/metabolism ; Wnt Signaling Pathway ; },
abstract = {The pluripotency of embryonic stem cells (ESCs) is maintained by core pluripotency transcription factors, cofactors and several signaling pathways. RBM14 is a component of the para-speckle complex, which has been implicated in multiple important biological processes. The role of RBM14 in ESCs and lineage differentiation remains to be elucidated. In the present study, we provided evidence that RBM14 plays important roles in maintaining pluripotency and in the early differentiation of ESCs. RBM14 was demonstrated to be expressed in mouse embryonic stem cells (mESCs) and localized in the nucleus. RBM14 expression was depleted in mESCs using clustered regularly interspaced short palindromic repeats (CRISPR) technology. Our results also showed that RBM14 depletion altered the gene expression profiles of mESCs. In particular, pluripotency-associated genes and genes involved in the Wnt and TGF-β signaling pathways were downregulated in RBM14 knockout mESCs. Furthermore, RBM14 was found to be essential for mesoderm development in vitro and in vivo. The specific effects of RBM14 depletion were verified by conducting a rescue experiment. Our findings demonstrated that RBM14 not only plays an important role in maintaining the pluripotency of mESCs but is also indispensable for mesoderm development.},
}

Animals
CRISPR-Cas Systems
Cell Differentiation
Gene Knockout Techniques
Mesoderm/*embryology/*metabolism
Mice
Mice, Inbred NOD
Mice, SCID
Mouse Embryonic Stem Cells/*cytology/*metabolism/transplantation
Transcription Factors/deficiency/genetics/*metabolism
Transcriptome
Transforming Growth Factor beta/metabolism
Wnt Signaling Pathway

@article {pmid29725684,
year = {2018},
author = {Liu, Y and Zhao, G and Xu, CF and Luo, YL and Lu, ZD and Wang, J},
title = {Systemic delivery of CRISPR/Cas9 with PEG-PLGA nanoparticles for chronic myeloid leukemia targeted therapy.},
journal = {Biomaterials science},
volume = {6},
number = {6},
pages = {1592-1603},
doi = {10.1039/c8bm00263k},
pmid = {29725684},
issn = {2047-4849},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Fusion Proteins, bcr-abl/*genetics ; Genetic Therapy/*methods ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*genetics/*therapy ; Mice, Inbred NOD ; Mice, SCID ; Nanoparticles/*chemistry ; Polyesters/*chemistry ; Polyethylene Glycols/*chemistry ; },
abstract = {Chronic myeloid leukemia (CML), which is characterized by the Philadelphia translocation, which fuses breakpoint cluster region (BCR) sequences from chromosome 22 upstream of the Abelson murine leukemia viral oncogene homolog (ABL) on chromosome 9, requires specific and efficient treatment. The CRISPR/Cas9 system, with its mechanism of specific DNA complementary recognition by engineered guide RNA (gRNA), allows the development of novel therapeutics for CML. To achieve targeted therapy of CML with the CRISPR/Cas9 system, we encapsulated a CRISPR/Cas9 plasmid (pCas9) expressing gRNA targeting the overhanging fusion region of the BCR-ABL gene (pCas9/gBCR-ABL) with poly(ethylene glycol)-b-poly(lactic acid-co-glycolic acid) (PEG-PLGA)-based cationic lipid-assisted polymeric nanoparticles (CLANs), which specifically disrupted the CML-related BCR-ABL gene while sparing the BCR and ABL genes in normal cells. After intravenous injection, CLANs carrying pCas9/gBCR-ABL (CLANpCas9/gBCR-ABL) efficiently knocked out the BCR-ABL fusion gene of CML cells and improved the survival of a CML mouse model, indicating that the combination of the CRISPR/Cas9 system with nanocarriers is a promising strategy for targeted treatment of CML.},
}

Animals
*CRISPR-Cas Systems
Cell Line, Tumor
Female
Fusion Proteins, bcr-abl/*genetics
Genetic Therapy/*methods
Humans
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*genetics/*therapy
Mice, Inbred NOD
Mice, SCID
Nanoparticles/*chemistry
Polyesters/*chemistry
Polyethylene Glycols/*chemistry

@article {pmid29567476,
year = {2018},
author = {Li, W and Zhang, G and Li, X and Wang, X and Li, Q and Hong, L and Shen, Y and Zhao, C and Gong, X and Chen, Y and Zhou, J},
title = {Thyroid hormone receptor interactor 13 (TRIP13) overexpression associated with tumor progression and poor prognosis in lung adenocarcinoma.},
journal = {Biochemical and biophysical research communications},
volume = {499},
number = {3},
pages = {416-424},
doi = {10.1016/j.bbrc.2018.03.129},
pmid = {29567476},
issn = {1090-2104},
mesh = {ATPases Associated with Diverse Cellular Activities/*genetics/metabolism ; Adenocarcinoma/*genetics/*pathology ; Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints/genetics ; Cell Cycle Proteins/*genetics/metabolism ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation ; *Disease Progression ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Gene Silencing ; Humans ; Lung Neoplasms/*genetics/*pathology ; Mice, Inbred BALB C ; Mice, Nude ; Phosphorylation ; Prognosis ; Protein Interaction Maps ; Proto-Oncogene Proteins c-akt/metabolism ; RNA, Guide/genetics/metabolism ; Survival Analysis ; Up-Regulation/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Thyroid hormone receptor interactor 13 (TRIP13) is an AAA+-ATPase that plays a key role in mitotic checkpoint complex inactivation and is associated with the progression of several cancers. However, its role in lung adenocarcinogenesis remains unknown. Here, we report that TRIP13 is highly overexpressed in multiple lung adenocarcinoma cell lines and tumor tissues. Clinically, TRIP13 expression is positively associated with tumor size, T-stage, and N-stage, and Kaplan-Meier analysis revealed that heightened TRIP13 expression is associated with lower overall survival. TRIP13 promotes lung adenocarcinoma cell proliferation, clonogenicity, and migration while inhibiting apoptosis and G2/M phase shift in vitro. Accordingly, TRIP13-silenced xenograft tumors displayed significant growth inhibition in vivo. Bioinformatics analysis demonstrated that TRIP13 interacts with a protein network associated with dsDNA break repair and PI3K/Akt signaling. TRIP13 upregulatesAktSer473 and downregulatesAktThr308/mTORSer2448activity, which suppresses accurate dsDNA break repair. TRIP13 also downregulates pro-apoptotic BadSer136 and cleaved caspase-3 while upregulating survivin. In conclusion, heightened TRIP13 expression appears to promote lung adenocarcinoma tumor progression and displays potential as a therapeutic target or biomarker for lung adenocarcinoma.},
}

ATPases Associated with Diverse Cellular Activities/*genetics/metabolism
Adenocarcinoma/*genetics/*pathology
Animals
Apoptosis/genetics
CRISPR-Cas Systems/genetics
Cell Cycle Checkpoints/genetics
Cell Cycle Proteins/*genetics/metabolism
Cell Line, Tumor
Cell Movement/genetics
Cell Proliferation
*Disease Progression
Down-Regulation
Female
Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Gene Silencing
Humans
Lung Neoplasms/*genetics/*pathology
Mice, Inbred BALB C
Mice, Nude
Phosphorylation
Prognosis
Protein Interaction Maps
Proto-Oncogene Proteins c-akt/metabolism
RNA, Guide/genetics/metabolism
Survival Analysis
Up-Regulation/genetics
Xenograft Model Antitumor Assays

@article {pmid29380513,
year = {2018},
author = {Jia, S and Zhang, R and Lin, G and Peng, R and Gao, P and Han, Y and Fu, Y and Ding, J and Wu, Q and Zhang, K and Xie, J and Li, J},
title = {A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing.},
journal = {Journal of clinical laboratory analysis},
volume = {32},
number = {5},
pages = {e22391},
doi = {10.1002/jcla.22391},
pmid = {29380513},
issn = {1098-2825},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Mutational Analysis ; Glycine/*genetics ; HEK293 Cells ; Humans ; Mutation/*genetics ; Proto-Oncogene Proteins p21(ras)/*genetics ; Transfection ; Valine/*genetics ; },
abstract = {BACKGROUND: KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations.

METHODS: We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS.

RESULTS: We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively.

CONCLUSION: The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.},
}

CRISPR-Cas Systems/*genetics
Cell Line
DNA Mutational Analysis
Glycine/*genetics
HEK293 Cells
Humans
Mutation/*genetics
Proto-Oncogene Proteins p21(ras)/*genetics
Transfection
Valine/*genetics

@article {pmid29150593,
year = {2018},
author = {Xie, ZX and Mitchell, LA and Liu, HM and Li, BZ and Liu, D and Agmon, N and Wu, Y and Li, X and Zhou, X and Li, B and Xiao, WH and Ding, MZ and Wang, Y and Yuan, YJ and Boeke, JD},
title = {Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {1},
pages = {173-183},
doi = {10.1534/g3.117.300347},
pmid = {29150593},
issn = {2160-1836},
mesh = {*CRISPR-Cas Systems ; Cell Engineering/methods ; Chromosomes, Artificial/chemistry ; DNA Breaks, Double-Stranded ; DNA, Fungal/*genetics/metabolism ; Gene Editing/*methods ; *Genes, Mating Type, Fungal ; Genetic Loci ; *Genome, Fungal ; Plasmids/chemistry/metabolism ; Ploidies ; RNA, Guide/genetics/metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; },
abstract = {Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ∼80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLα and HMRa cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully a/a/α/α isogenic tetraploids.},
}

*CRISPR-Cas Systems
Cell Engineering/methods
Chromosomes, Artificial/chemistry
DNA Breaks, Double-Stranded
DNA, Fungal/*genetics/metabolism
Gene Editing/*methods
*Genes, Mating Type, Fungal
Genetic Loci
*Genome, Fungal
Plasmids/chemistry/metabolism
Ploidies
RNA, Guide/genetics/metabolism
Saccharomyces cerevisiae/*genetics/metabolism

@article {pmid28439558,
year = {2017},
author = {Zhang, Y and Long, C and Li, H and McAnally, JR and Baskin, KK and Shelton, JM and Bassel-Duby, R and Olson, EN},
title = {CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice.},
journal = {Science advances},
volume = {3},
number = {4},
pages = {e1602814},
doi = {10.1126/sciadv.1602814},
pmid = {28439558},
issn = {2375-2548},
support = {R01 HL077439/HL/NHLBI NIH HHS/United States ; R01 DK099653/DK/NIDDK NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 HL111665/HL/NHLBI NIH HHS/United States ; R01 AR067294/AR/NIAMS NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Dystrophin/genetics/metabolism ; Humans ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Animal/genetics/metabolism/pathology/*therapy ; Muscular Dystrophy, Duchenne/genetics/metabolism/pathology/*therapy ; Myocytes, Cardiac/*metabolism/pathology ; },
abstract = {Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene (DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD.},
}

Animals
*CRISPR-Cas Systems
*Dystrophin/genetics/metabolism
Humans
Mice
Mice, Inbred mdx
Muscular Dystrophy, Animal/genetics/metabolism/pathology/*therapy
Muscular Dystrophy, Duchenne/genetics/metabolism/pathology/*therapy
Myocytes, Cardiac/*metabolism/pathology

@article {pmid28435878,
year = {2017},
author = {Noble, C and Olejarz, J and Esvelt, KM and Church, GM and Nowak, MA},
title = {Evolutionary dynamics of CRISPR gene drives.},
journal = {Science advances},
volume = {3},
number = {4},
pages = {e1601964},
doi = {10.1126/sciadv.1601964},
pmid = {28435878},
issn = {2375-2548},
mesh = {CRISPR-Cas Systems/*physiology ; *Evolution, Molecular ; *Models, Genetic ; },
abstract = {The alteration of wild populations has been discussed as a solution to a number of humanity's most pressing ecological and public health concerns. Enabled by the recent revolution in genome editing, clustered regularly interspaced short palindromic repeats (CRISPR) gene drives-selfish genetic elements that can spread through populations even if they confer no advantage to their host organism-are rapidly emerging as the most promising approach. However, before real-world applications are considered, it is imperative to develop a clear understanding of the outcomes of drive release in nature. Toward this aim, we mathematically study the evolutionary dynamics of CRISPR gene drives. We demonstrate that the emergence of drive-resistant alleles presents a major challenge to previously reported constructs, and we show that an alternative design that selects against resistant alleles could greatly improve evolutionary stability. We discuss all results in the context of CRISPR technology and provide insights that inform the engineering of practical gene drive systems.},
}

CRISPR-Cas Systems/*physiology
*Evolution, Molecular
*Models, Genetic

@article {pmid30295654,
year = {2018},
author = {Du, L and Zhou, A and Sohr, A and Roy, S},
title = {An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {139},
pages = {},
doi = {10.3791/58268},
pmid = {30295654},
issn = {1940-087X},
abstract = {Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a "Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated" (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.},
}

@article {pmid30295162,
year = {2018},
author = {Ali, Q},
title = {Non-conventional therapeutic technique to replace CRISPR bacteria from biofilm by inducible lysogen.},
journal = {Journal of biological dynamics},
volume = {},
number = {},
pages = {1-28},
doi = {10.1080/17513758.2018.1527958},
pmid = {30295162},
issn = {1751-3766},
abstract = {Bacteriophage can be an effective means of regulating bacterial populations when conditions allow phage invasion of bacterial colonies. Phage can either infect and lyse a host cell, or insert their DNA into the host cell genome; the latter process is called lysogeny. The clustered regularly interspaced short palindromic repeat (CRISPR) system, linked with CRISPR-associated (Cas) genes, is a regulatory system present in a variety of bacteria which confers immunity against bacteriophage. Studies of the group behaviour of bacteria with CRISPR/Cas systems have provided evidence that CRISPR in lysogenized bacteria can cause an inability to form biofilm. This allows CRISPR-immune bacteria in biofilms to effectively resist phage therapy. Our recent work has described a potential therapeutic technique to eradicate CRISPR-immune bacteria from a biofilm by a continuous influx of lysogens carrying an identical phage sequence. However, this model predicted that the CRISPR-immune population could persist for long times before eradication. Our current focus is on the use of diverse lysogens against CRISPR-capable bacterial populations. The goal of this work is to find a suitable strategy which can eradicate bacteria with a CRISPR system through the influx of finite amounts of distinct lysogens over fixed intervals.},
}

@article {pmid29844281,
year = {2018},
author = {Wang, X and Zheng, P and Ma, T and Song, T},
title = {Small Universal Bacteria and Plasmid Computing Systems.},
journal = {Molecules (Basel, Switzerland)},
volume = {23},
number = {6},
pages = {},
doi = {10.3390/molecules23061307},
pmid = {29844281},
issn = {1420-3049},
mesh = {Adenosine Triphosphate/metabolism ; *Algorithms ; Bacteria/*chemistry/genetics/metabolism ; CRISPR-Cas Systems ; Cell Engineering/methods/*statistics & numerical data ; Conjugation, Genetic ; Endonucleases/genetics/metabolism ; Gene Editing ; *Genome, Bacterial ; *Mathematical Computing ; Plasmids/*chemistry/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {Bacterial computing is a known candidate in natural computing, the aim being to construct "bacterial computers" for solving complex problems. In this paper, a new kind of bacterial computing system, named the bacteria and plasmid computing system (BP system), is proposed. We investigate the computational power of BP systems with finite numbers of bacteria and plasmids. Specifically, it is obtained in a constructive way that a BP system with 2 bacteria and 34 plasmids is Turing universal. The results provide a theoretical cornerstone to construct powerful bacterial computers and demonstrate a concept of paradigms using a "reasonable" number of bacteria and plasmids for such devices.},
}

Adenosine Triphosphate/metabolism
*Algorithms
Bacteria/*chemistry/genetics/metabolism
CRISPR-Cas Systems
Cell Engineering/methods/*statistics & numerical data
Conjugation, Genetic
Endonucleases/genetics/metabolism
Gene Editing
*Genome, Bacterial
*Mathematical Computing
Plasmids/*chemistry/metabolism
RNA, Guide/genetics/metabolism

@article {pmid30290202,
year = {2018},
author = {Chen, S and Liu, H and Liang, W and Hong, L and Zhang, B and Huang, L and Guo, X and Duan, G},
title = {Insert sequences of CRISPR/Cas system regulate horizontal antibiotic gene transfer in Shigella.},
journal = {International journal of antimicrobial agents},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ijantimicag.2018.09.020},
pmid = {30290202},
issn = {1872-7913},
abstract = {Multidrug resistances Shigella is an enormous threat of public health. The resistance genes always located in plasmids, phages and integrons, which get into bacteria cells by horizontal gene transfer (HGT). CRISPR-Cas systems are adaptive immune systems in bacteria that are beneficial to resist phage infection and other mobile genetic elements. But this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Here, we investigate how Shigella regulate the activity of the CRISPR/Cas system spontaneously, when it wants acquire exogenous gene that is necessary for its survival. We found that IS elements were identified in cas genes, such as IS600 in cse2, ISSfl2 in cas6e, IS629 in cse1- cas3. The number of spacers in CRISPR/Cas which strain containing IS was fewer compare with none IS. Interestingly, fewer spacers were also found in multi-drug resistance Shigella strains. Furthermore, we have constructed the genetic transferred antibiotic resistant strain by resistance plasmid transfer, to detect the CRISPR/Cas system changes in this two group strains. We found that cse2 gene had a new IS elements (IS600) in the antibiotic-resistant strain. The bioinformatics analyses showed that the IS600 insert hotspots in the cse2 gene were the TGC-GGC gene motifs and the tertiary structure of the Cse2 protein was different with IS600 or not. IS600 could bring a 5-order of magnitude (105) decrease in the relative expression of cse2 gene. This study has significant implication for further revealing the mechanism underlying the CRISPR/Cas-mediated antibiotic resistance gene horizontal transfer in Shigella.},
}

@article {pmid30289378,
year = {2018},
author = {Tashkandi, M and Ali, Z and Aljedaani, F and Shami, A and Mahfouz, MM},
title = {Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato.},
journal = {Plant signaling & behavior},
volume = {},
number = {},
pages = {1-7},
doi = {10.1080/15592324.2018.1525996},
pmid = {30289378},
issn = {1559-2324},
abstract = {CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.},
}

@article {pmid29888283,
year = {2018},
author = {Yasunaga, K and Ito, T and Miki, M and Ueda, K and Fujiyama, T and Tachibana, Y and Fujimori, N and Kawabe, K and Ogawa, Y},
title = {Using CRISPR/Cas9 to Knock out Amylase in Acinar Cells Decreases Pancreatitis-Induced Autophagy.},
journal = {BioMed research international},
volume = {2018},
number = {},
pages = {8719397},
doi = {10.1155/2018/8719397},
pmid = {29888283},
issn = {2314-6141},
mesh = {*Acinar Cells/metabolism/pathology ; Amylases/*genetics/metabolism ; Autophagy/*genetics ; Autophagy-Related Protein 12/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; *Gene Knockdown Techniques ; Humans ; *Neoplasm Proteins/genetics/metabolism ; *Pancreatic Neoplasms/genetics/metabolism/pathology ; *Pancreatitis/genetics/metabolism/pathology ; },
abstract = {Pancreatic cancer is a malignant neoplasm that originates from acinar cells. Acinar cells get reprogrammed to become duct cells, resulting in pancreatic cancer. Pancreatitis is an acinar cell inflammation, leading to "impaired autophagy flux". Pancreatitis promotes acinar-to-ductal transdifferentiation. Expression of amylase gets eliminated during the progression of pancreatic cancer. Amylase is considered as an acinar cell marker; however, its function in cells is not known. Thus, we investigated whether amylase affects the acinar cell autophagy and whether it plays any role in development of pancreatitis. Here, we knocked out ATG12 in a pancreatic cancer cells and acinar cells using CRISPR/Cas9. Autophagy inhibition led to an increase in the expression of duct cell markers and a simultaneous decrease in that of acinar cell markers. It also caused an increase in cell viability and changes in mitochondrial morphology. Next, we knocked out amylase in acinar cells. Amylase deficiency decreased autophagy induced by pancreatitis. Our results suggest that amylase controls pancreatitis-induced autophagy. We found that eliminating amylase expression contributes to pancreatic cancer etiology by decreasing autophagy. Furthermore, our results indicate that amylase plays a role in selective pancreatitis-induced autophagy of pancreatic enzyme vesicles.},
}

*Acinar Cells/metabolism/pathology
Amylases/*genetics/metabolism
Autophagy/*genetics
Autophagy-Related Protein 12/genetics/metabolism
*CRISPR-Cas Systems
Cell Line
*Gene Knockdown Techniques
Humans
*Neoplasm Proteins/genetics/metabolism
*Pancreatic Neoplasms/genetics/metabolism/pathology
*Pancreatitis/genetics/metabolism/pathology

@article {pmid29273627,
year = {2018},
author = {Nakajima, K and Zhou, Y and Tomita, A and Hirade, Y and Gurumurthy, CB and Nakada, S},
title = {Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair.},
journal = {Genome research},
volume = {28},
number = {2},
pages = {223-230},
doi = {10.1101/gr.226027.117},
pmid = {29273627},
issn = {1549-5469},
mesh = {CRISPR-Cas Systems/*genetics ; Carrier Proteins/genetics ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics ; Deoxyribonuclease I/genetics ; Gene Editing ; Genetic Engineering/methods ; Genome, Human/*genetics ; Humans ; INDEL Mutation/genetics ; Mutagenesis/genetics ; Nuclear Proteins/genetics ; Rad51 Recombinase/genetics ; Rad52 DNA Repair and Recombination Protein/genetics ; Recombinational DNA Repair/*genetics ; },
abstract = {CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.},
}

CRISPR-Cas Systems/*genetics
Carrier Proteins/genetics
*DNA Breaks, Double-Stranded
DNA End-Joining Repair/genetics
Deoxyribonuclease I/genetics
Gene Editing
Genetic Engineering/methods
Genome, Human/*genetics
Humans
INDEL Mutation/genetics
Mutagenesis/genetics
Nuclear Proteins/genetics
Rad51 Recombinase/genetics
Rad52 DNA Repair and Recombination Protein/genetics
Recombinational DNA Repair/*genetics

@article {pmid28753233,
year = {2018},
author = {Zuo, YY and Huang, JL and Wang, J and Feng, Y and Han, TT and Wu, YD and Yang, YH},
title = {Knockout of a P-glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua.},
journal = {Insect molecular biology},
volume = {27},
number = {1},
pages = {36-45},
doi = {10.1111/imb.12338},
pmid = {28753233},
issn = {1365-2583},
mesh = {ATP-Binding Cassette, Sub-Family B, Member 1/*genetics/metabolism ; Animals ; Bacillus thuringiensis/chemistry ; Bacterial Proteins/pharmacology ; CRISPR-Cas Systems ; Drug Resistance, Multiple/genetics ; Endotoxins/pharmacology ; Gene Knockout Techniques ; Hemolysin Proteins/pharmacology ; Insect Proteins/*genetics/metabolism ; Insecticide Resistance/*genetics ; Insecticides/*pharmacology ; Ivermectin/analogs & derivatives/pharmacology ; Larva/genetics/growth & development/physiology ; Spodoptera/genetics/growth & development/*physiology ; },
abstract = {P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta-cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua.},
}

ATP-Binding Cassette, Sub-Family B, Member 1/*genetics/metabolism
Animals
Bacillus thuringiensis/chemistry
Bacterial Proteins/pharmacology
CRISPR-Cas Systems
Drug Resistance, Multiple/genetics
Endotoxins/pharmacology
Gene Knockout Techniques
Hemolysin Proteins/pharmacology
Insect Proteins/*genetics/metabolism
Insecticide Resistance/*genetics
Insecticides/*pharmacology
Ivermectin/analogs & derivatives/pharmacology
Larva/genetics/growth & development/physiology
Spodoptera/genetics/growth & development/*physiology

@article {pmid28150375,
year = {2017},
author = {Li, XY and Liu, GC and Sheng, WJ and Dong, ZW and Chen, L and Zhao, RP and Wang, W},
title = {Genome editing in the butterfly type-species Papilio machaon.},
journal = {Insect science},
volume = {24},
number = {4},
pages = {708-711},
doi = {10.1111/1744-7917.12421},
pmid = {28150375},
issn = {1744-7917},
mesh = {Animals ; Base Sequence ; Butterflies/*genetics ; CRISPR-Cas Systems ; Gene Editing/*methods ; Insect Proteins/*genetics ; Mutagenesis ; Sequence Alignment ; },
}

Animals
Base Sequence
Butterflies/*genetics
CRISPR-Cas Systems
Gene Editing/*methods
Insect Proteins/*genetics
Mutagenesis
Sequence Alignment

@article {pmid30285540,
year = {2018},
author = {Smith, CIE and Zain, R},
title = {Therapeutic Oligonucleotides: State of the Art.},
journal = {Annual review of pharmacology and toxicology},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-pharmtox-010818-021050},
pmid = {30285540},
issn = {1545-4304},
abstract = {Oligonucleotides (ONs) can interfere with biomolecules representing the entire extended central dogma. Antisense gapmer, steric block, splice-switching ONs, and short interfering RNA drugs have been successfully developed. Moreover, antagomirs (antimicroRNAs), microRNA mimics, aptamers, DNA decoys, DNAzymes, synthetic guide strands for CRISPR/Cas, and innate immunity-stimulating ONs are all in clinical trials. DNA-targeting, triplex-forming ONs and strand-invading ONs have made their mark on drug development research, but not yet as medicines. Both design and synthetic nucleic acid chemistry are crucial for achieving biologically active ONs. The dominating modifications are phosphorothioate linkages, base methylation, and numerous 2' -substitutions in the furanose ring, such as 2' -fluoro, O-methyl, or methoxyethyl. Locked nucleic acid and constrained ethyl, a related variant, are bridged forms where the 2' -oxygen connects to the 4' -carbon in the sugar. Phosphorodiamidate morpholino oligomers, carrying a modified heterocyclic backbone ring, have also been commercialized. Delivery remains a major obstacle, but systemic administration and intrathecal infusion are used for treatment of the liver and brain, respectively. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 59 is January 6, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}

@article {pmid30284045,
year = {2018},
author = {Majumdar, S and Terns, MP},
title = {CRISPR RNA-guided DNA cleavage by reconstituted Type I-A immune effector complexes.},
journal = {Extremophiles : life under extreme conditions},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00792-018-1057-0},
pmid = {30284045},
issn = {1433-4909},
support = {R35GM118160//National Institutes of Health/ ; },
abstract = {Diverse CRISPR-Cas immune systems protect archaea and bacteria from viruses and other mobile genetic elements. All CRISPR-Cas systems ultimately function by sequence-specific destruction of invading complementary nucleic acids. However, each CRISPR system uses compositionally distinct crRNP [CRISPR (cr) RNA/Cas protein] immune effector complexes to recognize and destroy invasive nucleic acids by unique molecular mechanisms. Previously, we found that Type I-A (Csa) effector crRNPs from Pyrococcus furiosus function in vivo to eliminate invader DNA. Here, we reconstituted functional Type I-A effector crRNPs in vitro with recombinant Csa proteins and synthetic crRNA and characterized properties of crRNP assembly, target DNA recognition and cleavage. Six proteins (Csa 4-1, Cas3″, Cas3', Cas5a, Csa2, Csa5) are essential for selective target DNA binding and cleavage. Native gel shift analysis and UV-induced RNA-protein crosslinking demonstrate that Cas5a and Csa2 directly interact with crRNA 5' tag and guide sequences, respectively. Mutational analysis revealed that Cas3″ is the effector nuclease of the complex. Together, our results indicate that DNA cleavage by Type I-A crRNPs requires crRNA-guided and protospacer adjacent motif-dependent target DNA binding to unwind double-stranded DNA and expose single strands for progressive ATP-dependent 3'-5' cleavage catalyzed by integral Cas3' helicase and Cas3″ nuclease crRNP components.},
}

@article {pmid30283411,
year = {2018},
author = {Terceti, MS and Vences, A and Matanza, XM and Dalsgaard, I and Pedersen, K and Osorio, CR},
title = {Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity.},
journal = {Frontiers in microbiology},
volume = {9},
number = {},
pages = {2155},
doi = {10.3389/fmicb.2018.02155},
pmid = {30283411},
issn = {1664-302X},
abstract = {The marine bacterium Photobacterium damselae subsp. damselae is a pathogen for a variety of marine animals, as well as for humans, and is nowadays considered an emerging pathogen for fish of importance in marine aquaculture. Recent studies have suggested that outbreaks in fish farms are caused by multiclonal populations of this subspecies that exist in the environment. Here, we report the study of a collection of 31 strains isolated during the course of disease outbreaks in marine rainbow trout farms in Denmark in 1994, 1995, and 2006, respectively. A phylogenetic analysis based on the toxR gene sequence, and the screening of virulence-related genes uncovered a high genetic heterogeneity, even among strains isolated from the same fish farm at the same time. Moreover, comparative analysis of the whole genome sequences of four selected strains revealed a large number of differentially occurring genes, which included virulence genes, pPHDD1 plasmid, polysaccharide synthesis gene clusters, CRISPR-Cas systems and putative new mobile genetic elements. This study provides sound evidence that P. damselae subsp. damselae outbreaks in Danish rainbow trout farms were caused by multiclonal populations and that horizontal gene transfer constitutes a strong driving force in the generation of intraspecific diversity in this pathogen.},
}

@article {pmid30282704,
year = {2018},
author = {Hasan, MH and Davis, LE and Bollavarapu, RK and Mitra, D and Parmar, R and Tandon, R},
title = {Dynamin is required for efficient cytomegalovirus maturation and envelopment.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {},
doi = {10.1128/JVI.01418-18},
pmid = {30282704},
issn = {1098-5514},
abstract = {Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic assembly compartment (vAC) generated via a drastic reorganization of the membranes of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is implicated in membrane remodeling and endocytic membrane fission events; however, the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate early (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells as well as in mock-induced parental MEF at early times post infection although levels were reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels of IE1 protein and another viral early protein (m04) were normalized by 48 hours post infection; however, late protein (m55; gB) expression was reduced in infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact stages of nuclear virus maturation in both cases with equivalent numbers of nucleocapsids containing packaged viral DNA (C-capsids) indicating successful viral DNA replication, capsid assembly and genome packaging. Most importantly, severe defects in virus envelopment were visualized in TKO cells but not in parental cells. Dynamin inhibitor (dynasore) treated MEF showed a phenotype similar to TKO cells upon MCMV infection confirming the role of dynamin in late maturation processes. In summary, dynamin-mediated endocytic pathways are critical for the completion of cytoplasmic stages of cytomegalovirus maturation.ImportanceViruses are known to exploit specific cellular functions at different stages of their life cycle in order to replicate, avoid immune recognition by the host and to establish a successful infection. Cytomegalovirus (CMV) infected cells are characterized by a prominent cytoplasmic inclusion (virus assembly compartment; vAC) that is the site of virus maturation and envelopment. While endocytic membranes are known to be the functional components of vAC, knowledge of specific endocytic pathways implicated in CMV maturation and envelopment is lacking. Here we show that dynamin, which is an integral part of host endocytic machinery, is largely dispensable for early stages of CMV infection but is required at a late stage of CMV maturation. Studies on dynamin function in CMV infection will help us understand the host-virus interaction pathways amenable to targeting by conventional small molecules as well as by newer generation nucleotide-based therapeutics (e.g. siRNA, CRISPR/CAS gRNA, etc.).},
}

@article {pmid30150254,
year = {2018},
author = {Wang, Z and Hardcastle, TJ and Canto Pastor, A and Yip, WH and Tang, S and Baulcombe, DC},
title = {A novel DCL2-dependent miRNA pathway in tomato affects susceptibility to RNA viruses.},
journal = {Genes & development},
volume = {32},
number = {17-18},
pages = {1155-1160},
doi = {10.1101/gad.313601.118},
pmid = {30150254},
issn = {1549-5477},
mesh = {CRISPR-Cas Systems ; Disease Susceptibility ; Lycopersicon esculentum/enzymology/*genetics/*virology ; MicroRNAs/*metabolism ; Mutation ; Plant Diseases/virology ; RNA Viruses/*physiology ; RNA, Small Untranslated/metabolism ; Ribonuclease III/genetics/*metabolism ; },
abstract = {Tomato Dicer-like2 (slDCL2) is a key component of resistance pathways against potato virus X (PVX) and tobacco mosaic virus (TMV). It is also required for production of endogenous small RNAs, including miR6026 and other noncanonical microRNAs (miRNAs). The slDCL2 mRNAs are targets of these slDCL2-dependent RNAs in a feedback loop that was disrupted by target mimic RNAs of miR6026. In lines expressing these RNAs, there was correspondingly enhanced resistance against PVX and TMV. These findings illustrate a novel miRNA pathway in plants and a crop protection strategy in which miRNA target mimicry elevates expression of defense-related mRNAs.},
}

CRISPR-Cas Systems
Disease Susceptibility
Lycopersicon esculentum/enzymology/*genetics/*virology
MicroRNAs/*metabolism
Mutation
Plant Diseases/virology
RNA Viruses/*physiology
RNA, Small Untranslated/metabolism
Ribonuclease III/genetics/*metabolism

@article {pmid29241845,
year = {2018},
author = {Xue, WH and Xu, N and Yuan, XB and Chen, HH and Zhang, JL and Fu, SJ and Zhang, CX and Xu, HJ},
title = {CRISPR/Cas9-mediated knockout of two eye pigmentation genes in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).},
journal = {Insect biochemistry and molecular biology},
volume = {93},
number = {},
pages = {19-26},
doi = {10.1016/j.ibmb.2017.12.003},
pmid = {29241845},
issn = {1879-0240},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Compound Eye, Arthropod/*physiology ; Eye Color/genetics ; Female ; Gene Knockout Techniques ; Hemiptera/genetics/*physiology ; Insect Proteins/*genetics/metabolism ; Male ; *Pigmentation ; },
abstract = {The brown planthopper Nilaparvata lugens is one of the most destructive insect pests in Asia, demonstrating high fertility and causing huge crop losses by sucking sap of rice as well as transmitting viruses. However, functional genomic studies on N. lugens are seriously constrained by lack of genetic tools. Here, we employed two eye pigmentation genes to generate germ-line mutations in N. lugens using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system. We showed that injection of single guide RNA of the cinnabar gene of N. lugens (Nl-cn) into pre-blastoderm eggs induced insertion and deletion (indels) in the founder generation (G0), which were heritably transmitted to the following G1 generation, leading to bright red compound eyes and ocelli. Mutations of N. lugens white (Nl-w) generated a high mutant rate of up to 27.3%, resulting in mosaic eyes consisting of white and lightly pigmented ommatidia in both G0 and G1 individuals. The specificity of CRISPR/Cas9-mediated mutagenesis was further bolstered by PCR and RNA interference-based knockdown analysis. These results show that CRISPR/Cas9-mediated gene editing is achievable in a hemipteran insect, offering a valuable tool for the study of functional genomics and pest management in this planthopper species.},
}

Animals
Base Sequence
*CRISPR-Cas Systems
Compound Eye, Arthropod/*physiology
Eye Color/genetics
Female
Gene Knockout Techniques
Hemiptera/genetics/*physiology
Insect Proteins/*genetics/metabolism
Male
*Pigmentation

@article {pmid30278987,
year = {2018},
author = {Rui, Y and Wilson, DR and Green, JJ},
title = {Non-Viral Delivery To Enable Genome Editing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2018.08.010},
pmid = {30278987},
issn = {1879-3096},
abstract = {Genome-editing technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENS), and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biological research. Each biotechnology consists of a DNA-binding protein that can be programmed to recognize and initiate double-strand breaks (DSBs) for site-specific gene modification. These technologies have the potential to be harnessed to cure diseases caused by aberrant gene expression. To be successful therapeutically, their functionality depends on their safe and efficient delivery into the cell nucleus. This review discusses the challenges in the delivery of genome-editing tools, and highlights recent innovations in non-viral delivery that have potential to overcome these limitations and advance the translation of genome editing towards patient care.},
}

@article {pmid30139026,
year = {2018},
author = {Zuo, CX and Bian, XC and Yang, ZL and Feng, HL and Zhou, FY and Liu, YQ},
title = {[Establishment of Cas9 stably expressed human hepatocellular carcinoma and cholangiocarcinoma cell lines].},
journal = {Zhonghua zhong liu za zhi [Chinese journal of oncology]},
volume = {40},
number = {8},
pages = {572-579},
pmid = {30139026},
issn = {0253-3766},
support = {NSTI-CR17//National Science and Technology Infrastructure Program/ ; 2016-12M-3-005//CAMS Innovative Fund for Medical Science/ ; },
mesh = {Bile Duct Neoplasms/*genetics/pathology/virology ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; Carcinoma, Hepatocellular/*genetics/pathology/virology ; *Cell Line, Tumor/pathology/virology ; Cholangiocarcinoma/*genetics/pathology/virology ; Gallbladder Neoplasms/*genetics/pathology/virology ; Genetic Vectors ; Genome ; Humans ; Lentivirus ; Liver Neoplasms/*genetics/pathology/virology ; RNA, Guide ; },
abstract = {Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.},
}

Bile Duct Neoplasms/*genetics/pathology/virology
CRISPR-Associated Proteins/metabolism
CRISPR-Cas Systems/*genetics
Carcinoma, Hepatocellular/*genetics/pathology/virology
*Cell Line, Tumor/pathology/virology
Cholangiocarcinoma/*genetics/pathology/virology
Gallbladder Neoplasms/*genetics/pathology/virology
Genetic Vectors
Genome
Humans
Lentivirus
Liver Neoplasms/*genetics/pathology/virology
RNA, Guide

@article {pmid29626235,
year = {2018},
author = {Grzybek, M and Golonko, A and Górska, A and Szczepaniak, K and Strachecka, A and Lass, A and Lisowski, P},
title = {The CRISPR/Cas9 system sheds new lights on the biology of protozoan parasites.},
journal = {Applied microbiology and biotechnology},
volume = {102},
number = {11},
pages = {4629-4640},
doi = {10.1007/s00253-018-8927-3},
pmid = {29626235},
issn = {1432-0614},
support = {2012/07/N/NZ9/02060//Narodowe Centrum Nauki/ ; 2016/22/M/NZ2/00548//Narodowe Centrum Nauki/ ; 428/STYP/11/2016//Ministerstwo Nauki i Szkolnictwa Wyższego/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Eukaryota/genetics/*physiology ; Genome, Protozoan/genetics ; Parasites/genetics/*physiology ; },
abstract = {The CRISPR/Cas9 system, a natural defence system of bacterial organisms, has recently been used to modify genomes of the most important protozoa parasites. Successful genome manipulations with the CRISPR/Cas9 system are changing the present view of genetics in parasitology. The application of this system offers a major chance to overcome the current restriction in culturing, maintaining and analysing protozoan parasites, and allows dynamic analysis of parasite genes functions, leading to a better understanding of pathogenesis. CRISPR/Cas9 system will have a significant influence on the process of developing novel drugs and treatment strategies against protozoa parasites.},
}

Animals
*CRISPR-Cas Systems
Eukaryota/genetics/*physiology
Genome, Protozoan/genetics
Parasites/genetics/*physiology

@article {pmid29604449,
year = {2018},
author = {Zhai, J and Wang, Q and Gao, Y and Zhang, R and Li, S and Wei, B and You, Y and Sun, X and Lu, C},
title = {The mechanisms of Ag85A DNA vaccine activates RNA sensors through new signal transduction.},
journal = {International immunopharmacology},
volume = {59},
number = {},
pages = {1-11},
doi = {10.1016/j.intimp.2017.11.041},
pmid = {29604449},
issn = {1878-1705},
mesh = {Acyltransferases/*genetics ; Animals ; Antigens, Bacterial/*genetics ; CRISPR-Cas Systems ; Cell Line ; Dendritic Cells ; Female ; Interferon-beta/metabolism ; Mice, Inbred C57BL ; *RNA ; Signal Transduction ; *Vaccines, DNA ; },
abstract = {Low immunogenicity is one of the major problems limiting the clinical use for DNA vaccines, which makes it impossible to obtain a strong protective immune response after vaccination. In order to explore whether Ag85A DNA vaccine could mount more efficiently protective immune response through new RNA sensor and its signal transduction pathway of antigen presentation we designed and synthesized Ag85A gene fragment containing multiple points mutations and transfected the gene fragment into the dendritic cell line (DC2.4) by CRISPR/Cas9. Subsequently, we focused on the changes of RNA sensors RIG-I, Mda-5, and the downstream adaptors MAVS, IRF3, IRF7 and IFN-β. The results indicated the significant increases in the mRNA and protein expression of RNA sensors RIG-I, Mda-5 and related adaptors MAVS, IRF3, IRF7, and IFN-β in the mutant DC 2.4 cells. The flow cytometry results demonstrated that the expression of MHC II on the surface of DC 2.4 significantly increased when compared with that in control. Therefore, it is suggested that Ag85A mutant DNA could release immunogenic message through RNA sensors and related adaptors via non protein pathway. There is at least one RNA signal transduction pathway of Ag85A DNA in DC2.4 cell. The work provides a new mode of action for nucleic acid vaccine to improve immunogenicity and meaningful data for the better understanding of the mechanisms of DNA vaccine.},
}

Acyltransferases/*genetics
Animals
Antigens, Bacterial/*genetics
CRISPR-Cas Systems
Cell Line
Dendritic Cells
Female
Interferon-beta/metabolism
Mice, Inbred C57BL
*RNA
Signal Transduction
*Vaccines, DNA

@article {pmid28931646,
year = {2017},
author = {Madigan, NN and Staff, NP and Windebank, AJ and Benarroch, EE},
title = {Genome editing technologies and their potential to treat neurologic disease.},
journal = {Neurology},
volume = {89},
number = {16},
pages = {1739-1748},
doi = {10.1212/WNL.0000000000004558},
pmid = {28931646},
issn = {1526-632X},
mesh = {CRISPR-Cas Systems/genetics ; Child, Preschool ; *DNA Repair ; Dependovirus ; Female ; *Gene Editing ; *Genetic Therapy ; Genome, Human ; Humans ; Male ; Middle Aged ; Nervous System Diseases/diagnosis/*genetics/therapy ; },
}

CRISPR-Cas Systems/genetics
Child, Preschool
*DNA Repair
Dependovirus
Female
*Gene Editing
*Genetic Therapy
Genome, Human
Humans
Male
Middle Aged
Nervous System Diseases/diagnosis/*genetics/therapy

@article {pmid27412402,
year = {2016},
author = {Jackson, AA and Hinkley, TC and Talbert, JN and Nugen, SR and Sela, DA},
title = {Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli.},
journal = {The Analyst},
volume = {141},
number = {19},
pages = {5543-5548},
doi = {10.1039/c6an00479b},
pmid = {27412402},
issn = {1364-5528},
mesh = {Alkaline Phosphatase/*chemistry/genetics ; Bacteriophage T7/*genetics ; CRISPR-Cas Systems ; Escherichia coli/*isolation & purification ; Food Contamination/analysis ; *Genetic Engineering ; },
abstract = {A large fraction of foodborne illnesses are linked to (∼46%) leafy green vegetables contaminated by pathogens harbored in agricultural water. To prevent this, accurate point-of-production detection tools are required to identify and quantify bacterial contaminants in produce before consumers are impacted. In this study, a proof-of-concept model was engineered for a phage-based Escherichia coli detection system. We engineered the coliphage T7 to express alkaline phosphatase (ALP) to serve as the signal for E. coli detection. Wild type phoA (T7ALP) and a dominant-active allele, phoA D153G D330N (T7ALP*) was inserted into the T7 genome, with engineered constructs selected by CRISPR-mediated cleavage of unaltered chromosomes and confirmed by PCR. Engineered phages and E. coli target cells were co-incubated for 16 hours to produce lysates with liberated ALP correlated with input cell concentrations. A colorimetric assay used p-nitrophenyl phosphate (pNPP) to demonstrate significant ALP production by T7ALP and T7ALP* compared to the vector control (T7EV) (p≤ 0.05). Furthermore, T7ALP* produced 2.5-fold more signal than T7ALP (p≤ 0.05) at pH 10. Due to the increase in signal for the modified ALP* allele, we assessed T7ALP* sensitivity in a dose-responsive manner. We observed 3-fold higher signal for target cell populations as low as ∼2 × 10(5) CFU mL(-1) (p≤ 0.05 vs. no-phage control).},
}

Alkaline Phosphatase/*chemistry/genetics
Bacteriophage T7/*genetics
CRISPR-Cas Systems
Escherichia coli/*isolation & purification
Food Contamination/analysis
*Genetic Engineering

@article {pmid30277308,
year = {2018},
author = {Buerger, P and Weynberg, KD and Wood-Charlson, EM and Sato, Y and Willis, BL and van Oppen, MJH},
title = {Novel T4 bacteriophages associated with black band disease in corals.},
journal = {Environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1462-2920.14432},
pmid = {30277308},
issn = {1462-2920},
abstract = {Research into causative agents underlying coral disease have focused primarily on bacteria, whereas potential roles of viruses have been largely unaddressed. Bacteriophages may contribute to diseases through the lysogenic introduction of virulence genes into bacteria, or prevent diseases through lysis of bacterial pathogens. To identify candidate phages that may influence the pathogenicity of black band disease (BBD), communities of bacteria (16S rRNA) and T4-bacteriophages (gp23) were simultaneously profiled with amplicon sequencing among BBD-lesions and healthy-coral-tissue of Montipora hispida, as well as seawater (study site: the central Great Barrier Reef). Bacterial community compositions were distinct among BBD-lesions, healthy-coral-tissue, and seawater samples, as observed in previous studies. Surprisingly, however, viral beta diversities based on both operational taxonomic unit (OTU)-compositions and overall viral community compositions did not differ statistically between the BBD-lesions and healthy-coral-tissue. Nonetheless, relative abundances of three bacteriophage OTUs, affiliated to Cyanophage PRSM6 and Prochlorococcus phages P-SSM2, were significantly higher in BBD-lesions than in healthy tissue. In addition, 32 gp23 OTUs showed nucleotide similarities to existing CRISPR-Cas spacers in BBD associated cyanobacterial genomes. These OTUs associated with our BBD samples suggest the presence of bacteriophages that infect members of the cyanobacteria-dominated BBD community, and thus have potential roles in BBD pathogenicity. This article is protected by copyright. All rights reserved.},
}

@article {pmid30157762,
year = {2018},
author = {Butt, H and Jamil, M and Wang, JY and Al-Babili, S and Mahfouz, M},
title = {Engineering plant architecture via CRISPR/Cas9-mediated alteration of strigolactone biosynthesis.},
journal = {BMC plant biology},
volume = {18},
number = {1},
pages = {174},
doi = {10.1186/s12870-018-1387-1},
pmid = {30157762},
issn = {1471-2229},
support = {CRG4//King Abdullah University of Science and Technology/ ; },
mesh = {*CRISPR-Cas Systems ; Dioxygenases/*genetics/metabolism ; *Gene Expression Regulation, Plant ; Heterocyclic Compounds, 3-Ring/*metabolism ; Lactones/*metabolism ; Oryza/*genetics/metabolism ; Plant Growth Regulators/*metabolism ; Plant Proteins/*genetics/metabolism ; },
abstract = {BACKGROUND: Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering).

RESULTS: We used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a reduced height, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography-mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient.

CONCLUSIONS: Taken together, our results show the potential and feasibility of the use of the CRISPR/Cas9 system for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.},
}

*CRISPR-Cas Systems
Dioxygenases/*genetics/metabolism
*Gene Expression Regulation, Plant
Heterocyclic Compounds, 3-Ring/*metabolism
Lactones/*metabolism
Oryza/*genetics/metabolism
Plant Growth Regulators/*metabolism
Plant Proteins/*genetics/metabolism

@article {pmid30117250,
year = {2018},
author = {Cabello-Yeves, PJ and Picazo, A and Camacho, A and Callieri, C and Rosselli, R and Roda-Garcia, JJ and Coutinho, FH and Rodriguez-Valera, F},
title = {Ecological and genomic features of two widespread freshwater picocyanobacteria.},
journal = {Environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1462-2920.14377},
pmid = {30117250},
issn = {1462-2920},
support = {//FEDER funds/ ; //Generalitat Valenciana/ ; CGL2015-69557-R'//CLIMAWET/ ; //Spanish Ministry of Economy and Competitiveness/ ; //European Fund for Regional Development/ ; },
abstract = {We present two genomes of widespread freshwater picocyanobacteria isolated by extinction dilution from a Spanish oligotrophic reservoir. Based on microscopy and genomic properties, both picocyanobacteria were tentatively designated Synechococcus lacustris Tous, formerly described as a metagenome assembled genome (MAG) from the same habitat, and Cyanobium usitatum Tous, described here for the first time. Both strains were purified in unicyanobacterial cultures, and their genomes were sequenced. They are broadly distributed in freshwater systems; the first seems to be a specialist on temperate reservoirs (Tous, Amadorio, Dexter, Lake Lanier, Sparkling), and the second appears to also be abundant in cold environments including ice-covered lakes such as Lake Baikal, Lake Erie or the brackish Baltic Sea. Having complete genomes provided access to the flexible genome that does not assemble in MAGs. We found several genomic islands in both genomes, within which there were genes for nitrogen acquisition, transporters for a wide set of compounds and biosynthesis of phycobilisomes in both strains. Some of these regions of low coverage in metagenomes also included antimicrobial compounds, transposases and phage defence systems, including a novel type III CRISPR-Cas phage defence system that was only detected in Synechococcus lacustris Tous.},
}

@article {pmid30104361,
year = {2018},
author = {Zhang, Z and Niu, B and Ji, D and Li, M and Li, K and James, AA and Tan, A and Huang, Y},
title = {Silkworm genetic sexing through W chromosome-linked, targeted gene integration.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {35},
pages = {8752-8756},
doi = {10.1073/pnas.1810945115},
pmid = {30104361},
issn = {1091-6490},
mesh = {Animals ; Bombyx/*genetics ; *CRISPR-Cas Systems ; Chromosomes, Insect/*genetics ; Female ; Gene Editing/*methods ; Male ; Sex Chromosomes/*genetics ; *Sex Determination Processes ; },
abstract = {Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect Bombyx mori using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific nanos (nos) promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting transformer 2 (tra2), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for B. mori genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.},
}

Animals
Bombyx/*genetics
*CRISPR-Cas Systems
Chromosomes, Insect/*genetics
Female
Gene Editing/*methods
Male
Sex Chromosomes/*genetics
*Sex Determination Processes

@article {pmid29908341,
year = {2018},
author = {Ohde, T and Takehana, Y and Shiotsuki, T and Niimi, T},
title = {CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution.},
journal = {Arthropod structure & development},
volume = {47},
number = {4},
pages = {362-369},
doi = {10.1016/j.asd.2018.06.003},
pmid = {29908341},
issn = {1873-5495},
mesh = {Animals ; Base Sequence ; *Biological Evolution ; *CRISPR-Cas Systems ; Evolution, Molecular ; *Gene Expression Regulation, Developmental ; Insect Proteins/genetics ; Insecta/anatomy & histology/*genetics ; *Mutagenesis ; Phylogeny ; *Wings, Animal/anatomy & histology ; },
abstract = {Despite previous developmental studies on basally branching wingless insects and crustaceans, the evolutionary origin of insect wings remains controversial. Knowledge regarding genetic regulation of tissues hypothesized to have given rise to wings would help to elucidate how ancestral development changed to allow the evolution of true wings. However, genetic tools available for basally branching wingless species are limited. The firebrat Thermobia domestica is an apterygote species, phylogenetically related to winged insects. T. domestica presents a suitable morphology to investigate the origin of wings, as it forms the tergal paranotum, from which wings are hypothesized to have originated. Here we report the first successful CRISPR/Cas9-based germline genome editing in T. domestica. We provide a technological platform to understand the development of tissues hypothesized to have given rise to wings in an insect with a pre-wing evolution body plan.},
}

Animals
Base Sequence
*Biological Evolution
*CRISPR-Cas Systems
Evolution, Molecular
*Gene Expression Regulation, Developmental
Insect Proteins/genetics
Insecta/anatomy & histology/*genetics
*Mutagenesis
Phylogeny
*Wings, Animal/anatomy & histology

@article {pmid29890897,
year = {2018},
author = {Mudziwapasi, R and Ndudzo, A and Nyamusamba, RP and Jomane, FN and Mutengwa, TT and Maphosa, M},
title = {Unlocking the potential of CRISPR technology for improving livelihoods in Africa.},
journal = {Biotechnology & genetic engineering reviews},
volume = {34},
number = {2},
pages = {198-215},
doi = {10.1080/02648725.2018.1482101},
pmid = {29890897},
issn = {2046-5556},
mesh = {Africa ; Animals ; *CRISPR-Cas Systems ; Food Storage ; Food Supply ; Gene Editing/economics/*methods ; Humans ; Plants, Genetically Modified/*growth & development ; },
abstract = {Africa is burdened with food shortages and plant, animal and human diseases. Some of these can be ameliorated by adopting genome editing technologies such as CRISPR. This technology is considered better than its predecessors, Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), because it is cheaper, easy to use, has high gene modification efficiency and is less time consuming. CRISPR technology has wide applications in the African context ranging from crop and animal improvement to disease diagnosis and treatment as well as improving food shelf life, organoleptic properties and food safety. It has the potential to bring back species of organisms that are extinct. However, some African countries have not taken advantage of the potential of CRISPR to solve many of their problems. This paper explores possible applications of CRISPR towards improvement of African livelihoods.},
}

Africa
Animals
*CRISPR-Cas Systems
Food Storage
Food Supply
Gene Editing/economics/*methods
Humans
Plants, Genetically Modified/*growth & development

@article {pmid29746102,
year = {2018},
author = {Kartje, ZJ and Barkau, CL and Rohilla, KJ and Ageely, EA and Gagnon, KT},
title = {Chimeric Guides Probe and Enhance Cas9 Biochemical Activity.},
journal = {Biochemistry},
volume = {57},
number = {21},
pages = {3027-3031},
doi = {10.1021/acs.biochem.8b00107},
pmid = {29746102},
issn = {1520-4995},
mesh = {Bacterial Proteins/*chemistry/*genetics/metabolism ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics ; DNA Cleavage ; Endonucleases/*chemistry/*genetics/metabolism ; RNA, Bacterial/chemistry ; RNA, Guide ; },
abstract = {DNA substitutions in RNA can probe the importance of A-form structure, 2'-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. Using this approach, we found that Cas9 biochemical activity tolerated significant substitution with DNA nucleotides in the clustered regularly interspaced short palindromic repeat RNA (crRNA). Only minimal RNA content was needed in or near the seed region. Simultaneous substitution at all positions with predicted crRNA-Cas9 2'-hydroxyl contacts had no effect on enzyme activity. The trans-activating crRNA (tracrRNA) also tolerated >50% substitution with DNA. DNA substitutions in the tracrRNA-pairing region of crRNA consistently enhanced cleavage activity while maintaining or improving target specificity. Together, results point to a prominent role for guide:target A-form-like helical structure and a possible regulatory role for the crRNA-tracrRNA pairing motif. A model chimeric crRNA with high activity did not significantly alter RNP assembly or target binding but did reduce Cas9 ribonucleoprotein stability, suggesting effects through conformation or dynamics. Cas9 directed by chimeric RNA-DNA guides may represent a cost-effective synthetic or molecular biology tool for robust and specific DNA cleavage.},
}

Bacterial Proteins/*chemistry/*genetics/metabolism
CRISPR-Associated Proteins/metabolism
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA/genetics
DNA Cleavage
Endonucleases/*chemistry/*genetics/metabolism
RNA, Bacterial/chemistry
RNA, Guide

@article {pmid29716628,
year = {2018},
author = {Ling, K and Jiang, L and Liang, S and Kwong, J and Yang, L and Li, Y and PingYin, and Deng, Q and Liang, Z},
title = {Nanog interaction with the androgen receptor signaling axis induce ovarian cancer stem cell regulation: studies based on the CRISPR/Cas9 system.},
journal = {Journal of ovarian research},
volume = {11},
number = {1},
pages = {36},
doi = {10.1186/s13048-018-0403-2},
pmid = {29716628},
issn = {1757-2215},
support = {81602282//National Natural Science Foundation of China/ ; 81501264//National Natural Science Foundation of China/ ; CYB16121//The Postgraduate Scientific Innovation Project of Chongqing Education Commission/ ; },
mesh = {Androgens/genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Nanog Homeobox Protein/*genetics ; Neoplastic Stem Cells/pathology ; Ovarian Neoplasms/*genetics/pathology ; Ovary/growth & development/pathology ; Promoter Regions, Genetic/genetics ; Receptors, Androgen/*genetics ; Signal Transduction/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Ovarian cancer stem cells (OCSCs) contribute to the poor prognosis of ovarian cancer. Involvement of the androgen receptor (AR) in the malignant behaviors of other tumors has been reported. However, whether AR associates with Nanog (a stem cell marker) and participates in OCSC functions remain unclear. In this study, we investigated the interaction of Nanog with AR and examined whether this interaction induced stem-like properties in ovarian cancer cells.

METHODS: AR and Nanog expression in ovarian tumors was evaluated. Using the CRISPR/Cas9 system, we constructed a Nanog green fluorescent protein (GFP) marker cell model to investigate the expression and co-localization of Nanog and AR. Then, we examined the effect of androgen on the Nanog promoter in ovarian cancer cell lines (A2780 and SKOV3). After androgen or anti-androgen treatment, cell proliferation, migration, sphere formation, colony formation and tumorigenesis were assessed in vitro and in vivo.

RESULTS: Both AR and Nanog expression were obviously high in ovarian tumors. Our results showed that Nanog expression was correlated with AR expression. The androgen 5α-dihydrotestosterone (DHT) activated Nanog promoter transcription. Meanwhile, Nanog GFP-positive cells treated with DHT exhibited higher levels of proliferation, migration, sphere formation and colony formation. We also observed that the tumorigenesis of Nanog GFP-positive cells was significantly higher than that of the GFP-negative cells. Xenografts of Nanog GFP-positive cells showed significant differences when treated with androgen or anti-androgen drugs in vivo.

CONCLUSIONS: The interaction of Nanog with the AR signaling axis might induce or contribute to OCSC regulation. In addition, androgen might promote stemness characteristics in ovarian cancer cells by activating the Nanog promoter. This finding merits further study because it may provide a new understanding of OCSC regulation from a hormone perspective and lead to the reevaluation of stem cell therapy for ovarian cancer.},
}

Androgens/genetics/metabolism
Animals
CRISPR-Cas Systems/genetics
Carcinogenesis/*genetics
Cell Line, Tumor
Cell Movement
Cell Proliferation/genetics
Female
Gene Expression Regulation, Neoplastic
Humans
Mice
Nanog Homeobox Protein/*genetics
Neoplastic Stem Cells/pathology
Ovarian Neoplasms/*genetics/pathology
Ovary/growth & development/pathology
Promoter Regions, Genetic/genetics
Receptors, Androgen/*genetics
Signal Transduction/genetics
Xenograft Model Antitumor Assays

@article {pmid29685168,
year = {2018},
author = {Zhao, Q and Qian, Q and Cao, D and Yang, J and Gui, T and Shen, K},
title = {Role of BMI1 in epithelial ovarian cancer: investigated via the CRISPR/Cas9 system and RNA sequencing.},
journal = {Journal of ovarian research},
volume = {11},
number = {1},
pages = {31},
doi = {10.1186/s13048-018-0406-z},
pmid = {29685168},
issn = {1757-2215},
support = {81372780//National Natural Science Foundation of China (CN)/ ; 81402140//National Natural Science Foundation of China (CN)/ ; },
mesh = {Animals ; Apoptosis ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/genetics ; Flow Cytometry ; Focal Adhesions/genetics ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Mice ; Neoplasm Invasiveness/genetics ; Neoplasms, Glandular and Epithelial/*genetics/pathology ; Ovarian Neoplasms/*genetics/pathology ; Polycomb Repressive Complex 1/antagonists & inhibitors/*genetics ; Sequence Analysis, RNA ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) might be an appropriate biomarker in the management of epithelial ovarian cancer (EOC). However, the biological role of BMI1 and its relevant molecular mechanism needs further elaboration. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an excellent genome-editing tool and is scarcely used in EOC studies.

METHODS: We first applied CRISPR/Cas9 technique to silence BMI1 in EOC cells; thereafter we accomplished various in vivo and in vitro experiments to detect biological behaviors of ovarian cancer cells, including MTT, flow cytometry, Transwell, real-time polymerase chain reaction and western blotting assays, etc.; eventually, we used RNA sequencing to reveal the underlying molecular traits driven by BMI1 in EOC.

RESULTS: We successfully shut off the expression of BMI1 in EOC cells using CRISPR/Cas9 system, providing an ideal cellular model for investigations of target gene. Silencing BMI1 could reduce cell growth and metastasis, promote cell apoptosis, and enhance the platinum sensitivity of EOC cells. BMI1 might alter extracellular matrix structure and angiogenesis of tumor cells through regulating Focal adhesion and PI3K/AKT pathways.

CONCLUSION: BMI1 is a potential biomarker in EOC management, especially for tumor progression and chemo-resistance. Molecular traits, including BMI1 and core genes in Focal adhesion and PI3K/AKT pathways, might be alternatives as therapeutic targets for EOC.},
}

Animals
Apoptosis
*CRISPR-Cas Systems
Cell Line, Tumor
Cell Movement/genetics
Flow Cytometry
Focal Adhesions/genetics
Gene Expression Regulation, Neoplastic
Gene Silencing
High-Throughput Nucleotide Sequencing/*methods
Humans
Mice
Neoplasm Invasiveness/genetics
Neoplasms, Glandular and Epithelial/*genetics/pathology
Ovarian Neoplasms/*genetics/pathology
Polycomb Repressive Complex 1/antagonists & inhibitors/*genetics
Sequence Analysis, RNA
Xenograft Model Antitumor Assays

@article {pmid29487145,
year = {2018},
author = {Heigwer, F and Port, F and Boutros, M},
title = {RNA Interference (RNAi) Screening in Drosophila.},
journal = {Genetics},
volume = {208},
number = {3},
pages = {853-874},
doi = {10.1534/genetics.117.300077},
pmid = {29487145},
issn = {1943-2631},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Computational Biology/methods ; Drosophila/*genetics ; *Genetic Testing/methods ; Genome ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; *RNA Interference ; *RNA, Small Interfering ; Single-Cell Analysis ; Software ; },
abstract = {In the last decade, RNA interference (RNAi), a cellular mechanism that uses RNA-guided degradation of messenger RNA transcripts, has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function in Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. We will discuss available reagent libraries and cell lines, methodological approaches for cell-based assays, and computational methods for the analysis of high-throughput screens. Furthermore, we will review the generation and use of genome-scale RNAi libraries for tissue-specific knockdown analysis in vivo and discuss the differences and similarities with the use of genome-engineering methods such as CRISPR/Cas9 for functional analysis.},
}

Animals
CRISPR-Cas Systems
Cell Line
Computational Biology/methods
Drosophila/*genetics
*Genetic Testing/methods
Genome
Genomics/methods
High-Throughput Nucleotide Sequencing
*RNA Interference
*RNA, Small Interfering
Single-Cell Analysis
Software

@article {pmid29445053,
year = {2018},
author = {Leong, SY and Yamada, M and Yanagisawa, N and Goshima, G},
title = {SPIRAL2 Stabilises Endoplasmic Microtubule Minus Ends in the Moss Physcomitrella patens.},
journal = {Cell structure and function},
volume = {43},
number = {1},
pages = {53-60},
doi = {10.1247/csf.18001},
pmid = {29445053},
issn = {1347-3700},
mesh = {Bryopsida/*metabolism ; CRISPR-Cas Systems/genetics ; Frameshift Mutation ; Gene Deletion ; Gene Editing ; Genetic Loci ; Microtubule-Associated Proteins/classification/genetics/*metabolism ; Microtubules/*metabolism ; Phylogeny ; Plant Proteins/classification/genetics/*metabolism ; },
abstract = {Stabilisation of minus ends of microtubules (MTs) is critical for organising MT networks in land plant cells, in which all MTs are nucleated independent of centrosomes. Recently, Arabidopsis SPIRAL2 (SPR2) protein was shown to localise to plus and minus ends of cortical MTs, and increase stability of both ends. Here, we report molecular and functional characterisation of SPR2 of the basal land plant, the moss Physcomitrella patens. In protonemal cells of P. patens, where non-cortical, endoplasmic MT network is organised, we observed SPR2 at minus ends, but not plus ends, of endoplasmic MTs and likely also of phragmoplast MTs. Minus end decoration was reconstituted in vitro using purified SPR2, suggesting that moss SPR2 is a minus end-specific binding protein (-TIP). We generated a loss-of-function mutant of SPR2, in which frameshift-causing deletions/insertions were introduced into all four paralogous SPR2 genes by means of CRISPR/Cas9. Protonemal cells of the mutant showed instability of endoplasmic MT minus ends. These results indicate that moss SPR2 is a MT minus end stabilising factor.Key words: acentrosomal microtubule network, microtubule minus end, P. patens, CAMSAP/Nezha/Patronin.},
}

Bryopsida/*metabolism
CRISPR-Cas Systems/genetics
Frameshift Mutation
Gene Deletion
Gene Editing
Genetic Loci
Microtubule-Associated Proteins/classification/genetics/*metabolism
Microtubules/*metabolism
Phylogeny
Plant Proteins/classification/genetics/*metabolism

@article {pmid29444806,
year = {2018},
author = {Fan, Y and Lin, X},
title = {Multiple Applications of a Transient CRISPR-Cas9 Coupled with Electroporation (TRACE) System in the Cryptococcus neoformans Species Complex.},
journal = {Genetics},
volume = {208},
number = {4},
pages = {1357-1372},
doi = {10.1534/genetics.117.300656},
pmid = {29444806},
issn = {1943-2631},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cryptococcus neoformans/*genetics/metabolism ; *Electroporation ; Gene Deletion ; Gene Editing ; Gene Expression ; Gene Order ; Gene Targeting ; Genetic Vectors/genetics ; Homologous Recombination ; RNA, Guide ; Research ; Transformation, Genetic ; },
abstract = {Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the Cryptococcus genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (∼1-10%) are low. Here, we developed a Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation (TRACE) system for targeted genetic manipulations in the C. neoformans species complex. This method took advantages of efficient genome integration due to double-strand breaks created at specific sites by the transient CRISPR-Cas9 system and the high transformation efficiency of electroporation. We demonstrated that TRACE can efficiently generate precise single-gene deletion mutants using the ADE2 locus as an example. This system can also effectively delete multiple genes in a single transformation, as evident by the successful generation of quadruple mfα1Δ2Δ3Δ4Δ mutants. In addition to generating gene deletion mutants, we complemented the ade2Δ mutant by integrating a wild-type ADE2 allele at the "safe haven" region (SH2) via homologous recombination using TRACE. Interestingly, introduced DNAs can be inserted at a designated genetic site without any homologous sequences, opening up numerous other applications. We expect that TRACE, an efficient, versatile, and cost-effective gene editing approach, will greatly accelerate research in this field.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Cryptococcus neoformans/*genetics/metabolism
*Electroporation
Gene Deletion
Gene Editing
Gene Expression
Gene Order
Gene Targeting
Genetic Vectors/genetics
Homologous Recombination
RNA, Guide
Research
Transformation, Genetic

@article {pmid29348144,
year = {2018},
author = {Heppert, JK and Pani, AM and Roberts, AM and Dickinson, DJ and Goldstein, B},
title = {A CRISPR Tagging-Based Screen Reveals Localized Players in Wnt-Directed Asymmetric Cell Division.},
journal = {Genetics},
volume = {208},
number = {3},
pages = {1147-1164},
doi = {10.1534/genetics.117.300487},
pmid = {29348144},
issn = {1943-2631},
support = {R01 GM083071/GM/NIGMS NIH HHS/United States ; F32 GM115151/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; T32 CA009156/CA/NCI NIH HHS/United States ; K99 GM115964/GM/NIGMS NIH HHS/United States ; U41 HG002223/HG/NHGRI NIH HHS/United States ; T32 GM119999/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Asymmetric Cell Division/*genetics ; Biomarkers ; *CRISPR-Cas Systems ; Caenorhabditis elegans/genetics/metabolism ; Caenorhabditis elegans Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Transport ; Signal Transduction ; Spindle Apparatus/metabolism ; Stem Cells/metabolism ; Wnt Proteins/*metabolism ; },
abstract = {Oriented cell divisions are critical to establish and maintain cell fates and tissue organization. Diverse extracellular and intracellular cues have been shown to provide spatial information for mitotic spindle positioning; however, the molecular mechanisms by which extracellular signals communicate with cells to direct mitotic spindle positioning are largely unknown. In animal cells, oriented cell divisions are often achieved by the localization of force-generating motor protein complexes to discrete cortical domains. Disrupting either these force-generating complexes or proteins that globally affect microtubule stability results in defects in mitotic positioning, irrespective of whether these proteins function as spatial cues for spindle orientation. This poses a challenge to traditional genetic dissection of this process. Therefore, as an alternative strategy to identify key proteins that act downstream of intercellular signaling, we screened the localization of many candidate proteins by inserting fluorescent tags directly into endogenous gene loci, without overexpressing the proteins. We tagged 23 candidate proteins in Caenorhabditis elegans and examined each protein's localization in a well-characterized, oriented cell division in the four-cell-stage embryo. We used cell manipulations and genetic experiments to determine which cells harbor key localized proteins and which signals direct these localizations in vivo We found that Dishevelled and adenomatous polyposis coli homologs are polarized during this oriented cell division in response to a Wnt signal, but two proteins typically associated with mitotic spindle positioning, homologs of NuMA and Dynein, were not detectably polarized. These results suggest an unexpected mechanism for mitotic spindle positioning in this system, they pinpoint key proteins of interest, and they highlight the utility of a screening approach based on analyzing the localization of endogenously tagged proteins.},
}

Animals
Asymmetric Cell Division/*genetics
Biomarkers
*CRISPR-Cas Systems
Caenorhabditis elegans/genetics/metabolism
Caenorhabditis elegans Proteins/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Protein Transport
Signal Transduction
Spindle Apparatus/metabolism
Stem Cells/metabolism
Wnt Proteins/*metabolism

@article {pmid29301949,
year = {2018},
author = {Boateng, R and Allen, AK},
title = {New Role for an Old Protein: An Educational Primer for Use with "The Identification of a Novel Mutant Allele of topoisomerase II in Caenorhabditis elegans Reveals a Unique Role in Chromosome Segregation During Spermatogenesis".},
journal = {Genetics},
volume = {208},
number = {1},
pages = {79-88},
doi = {10.1534/genetics.117.300482},
pmid = {29301949},
issn = {1943-2631},
support = {R15 HD084253/HD/NICHD NIH HHS/United States ; },
mesh = {*Alleles ; Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/*metabolism ; Caenorhabditis elegans Proteins/genetics/metabolism ; *Chromosome Segregation ; DNA Topoisomerases/genetics/metabolism ; DNA Topoisomerases, Type II/*genetics/*metabolism ; Gene Editing ; Gene Expression Regulation, Developmental ; Germ Cells ; Miosis/genetics ; *Mutation ; Oogenesis/genetics ; RNA Interference ; Spermatogenesis/*genetics ; Whole Genome Sequencing ; },
abstract = {Modern experimental techniques, such as whole-genome sequencing and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endogenous genome editing, are enabling researchers to identify and further characterize the roles of proteins that were previously thought of as well defined. In the December 2016 issue of GENETICS, an article by Jaramillo-Lambert et al. identified a new role for the enzyme topoisomerase II in Caenorhabditis elegans male meiosis. This Primer article is designed to provide essential background information on C. elegans spermatogenesis and the relevant scientific techniques that will assist students and instructors in their understanding and discussion of the related article.Related article in GENETICS: Jaramillo-Lambert, A., A. S. Fabritius A. S., T. J. Hansen T. J., H. E. Smith H. E., and A. Golden A., 2016 The identification of a novel mutant allele of topoisomerase II in Caenorhabditis elegans reveals a unique role in chromosome segregation during spermatogenesis. Genetics204: 1407-1422.},
}

*Alleles
Animals
CRISPR-Cas Systems
Caenorhabditis elegans/*genetics/*metabolism
Caenorhabditis elegans Proteins/genetics/metabolism
*Chromosome Segregation
DNA Topoisomerases/genetics/metabolism
DNA Topoisomerases, Type II/*genetics/*metabolism
Gene Editing
Gene Expression Regulation, Developmental
Germ Cells
Miosis/genetics
*Mutation
Oogenesis/genetics
RNA Interference
Spermatogenesis/*genetics
Whole Genome Sequencing

@article {pmid29301908,
year = {2018},
author = {Johnson, KR and Gagnon, LH and Tian, C and Longo-Guess, CM and Low, BE and Wiles, MV and Kiernan, AE},
title = {Deletion of a Long-Range Dlx5 Enhancer Disrupts Inner Ear Development in Mice.},
journal = {Genetics},
volume = {208},
number = {3},
pages = {1165-1179},
doi = {10.1534/genetics.117.300447},
pmid = {29301908},
issn = {1943-2631},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 DC004301/DC/NIDCD NIH HHS/United States ; R01 DC009250/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Chromosome Mapping ; Chromosomes, Human, Pair 7 ; Ear, Inner/embryology/*metabolism/ultrastructure ; *Enhancer Elements, Genetic ; Female ; Genotype ; Heterozygote ; Homeodomain Proteins/*genetics ; Humans ; Mice ; Mice, Knockout ; Mutation ; Phenotype ; Proteasome Endopeptidase Complex/genetics ; Sequence Analysis, DNA ; *Sequence Deletion ; },
}

Animals
CRISPR-Cas Systems
Chromosome Mapping
Chromosomes, Human, Pair 7
Ear, Inner/embryology/*metabolism/ultrastructure
*Enhancer Elements, Genetic
Female
Genotype
Heterozygote
Homeodomain Proteins/*genetics
Humans
Mice
Mice, Knockout
Mutation
Phenotype
Proteasome Endopeptidase Complex/genetics
Sequence Analysis, DNA
*Sequence Deletion

@article {pmid29263213,
year = {2018},
author = {Xu, Z and Chang, CC and Li, M and Zhang, QY and Vasilescu, EM and D'Agati, V and Floratos, A and Vlad, G and Suciu-Foca, N},
title = {ILT3.Fc-CD166 Interaction Induces Inactivation of p70 S6 Kinase and Inhibits Tumor Cell Growth.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {200},
number = {3},
pages = {1207-1219},
doi = {10.4049/jimmunol.1700553},
pmid = {29263213},
issn = {1550-6606},
mesh = {Activated-Leukocyte Cell Adhesion Molecule/genetics/*metabolism ; Animals ; Antibodies, Monoclonal/immunology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/*genetics ; Dendritic Cells/immunology ; Female ; Gene Knockout Techniques ; Humans ; Immunoglobulin Fc Fragments/*immunology ; Jurkat Cells ; Mice ; Mice, Inbred NOD ; Neoplasms/*pathology ; Receptors, Cell Surface/*metabolism ; Ribosomal Protein S6 Kinases, 70-kDa/*metabolism ; Signal Transduction/genetics/immunology ; },
abstract = {The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4+ Th cell proliferation and to induce the generation of CD8+CD28- T suppressor cells. CD166 displays strong heterophilic interaction with CD6 and weaker homophilic CD166-CD166 cell adhesion interaction. ILT3.Fc inhibited the growth of CD166+ tumor cell lines (TCL) derived from lymphoid malignancies in vitro and in vivo. CRISPR-Cas9-based knockout of CD166 from TCL abrogated ILT3.Fc binding and its tumor-inhibitory effect. The mechanism underlying the effect of ILT3.Fc on tumor cell growth involves inhibition of the p70S6K signaling pathway. Blockade of CD166 by ILT3.Fc inhibited progression of human TCL in NOD.Cg-Prkdc Il-2rg/SzJ mice, suggesting its potential immunotherapeutic value.},
}

Activated-Leukocyte Cell Adhesion Molecule/genetics/*metabolism
Animals
Antibodies, Monoclonal/immunology
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cell Proliferation/*genetics
Dendritic Cells/immunology
Female
Gene Knockout Techniques
Humans
Immunoglobulin Fc Fragments/*immunology
Jurkat Cells
Mice
Mice, Inbred NOD
Neoplasms/*pathology
Receptors, Cell Surface/*metabolism
Ribosomal Protein S6 Kinases, 70-kDa/*metabolism
Signal Transduction/genetics/immunology

@article {pmid29235474,
year = {2017},
author = {Liu, Y and Han, J and Chen, Z and Wu, H and Dong, H and Nie, G},
title = {Engineering cell signaling using tunable CRISPR-Cpf1-based transcription factors.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {2095},
doi = {10.1038/s41467-017-02265-x},
pmid = {29235474},
issn = {2041-1723},
mesh = {Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Engineering/*methods ; Endonucleases/*genetics/metabolism ; Gene Editing/methods ; Gene Expression Regulation ; HEK293 Cells ; HeLa Cells ; Humans ; Receptors, G-Protein-Coupled/genetics/metabolism ; Signal Transduction/*genetics ; Transcription Factors/*genetics/metabolism ; },
abstract = {The catalytically dead Cpf1 endonuclease from Acidaminococcus sp. BV3L6 (dAsCpf1) has been used to construct effective transcriptional repressors in bacteria and plants. However, it is still unclear if dAsCpf1 can function in human cells as a transcriptional regulator or a signal conductor. Here, we repurpose the dAsCpf1 system in human cells for a variety of functions, including the activation or repression of gene transcription. Moreover, we construct programmable ligand-controlled dAsCpf1 systems either by coupling crRNAs with engineered riboswitches or by fusing dAsCpf1 proteins with G protein-coupled receptors. These generalizable approaches allow us to regulate the transcription of endogenous genes in response to diverse classes of ligands, thus constructing artificial signaling pathways with rewired cellular input-output behaviors. The systems exhibit signal amplification, an important feature in cell signaling, when multiple crRNAs are processed from a single transcript. The results provide a robust and efficient platform for engineering customized cell signaling circuits.},
}

Bacterial Proteins/*genetics/metabolism
*CRISPR-Cas Systems
Cell Engineering/*methods
Endonucleases/*genetics/metabolism
Gene Editing/methods
Gene Expression Regulation
HEK293 Cells
HeLa Cells
Humans
Receptors, G-Protein-Coupled/genetics/metabolism
Signal Transduction/*genetics
Transcription Factors/*genetics/metabolism

@article {pmid29235471,
year = {2017},
author = {Beishline, K and Vladimirova, O and Tutton, S and Wang, Z and Deng, Z and Lieberman, PM},
title = {CTCF driven TERRA transcription facilitates completion of telomere DNA replication.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {2114},
doi = {10.1038/s41467-017-02212-w},
pmid = {29235471},
issn = {2041-1723},
support = {P30 CA010815/CA/NCI NIH HHS/United States ; R01 CA140652/CA/NCI NIH HHS/United States ; T32 CA009171/CA/NCI NIH HHS/United States ; },
mesh = {Base Sequence ; CCCTC-Binding Factor/*genetics/metabolism ; CRISPR-Cas Systems ; *DNA Replication ; DNA-Binding Proteins/*genetics/metabolism ; Gene Editing/methods ; HCT116 Cells ; Humans ; Mutation ; Protein Binding ; Telomere/*genetics/metabolism ; Transcription Factors/*genetics/metabolism ; *Transcription, Genetic ; },
abstract = {Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres. Under replication stress, telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei. Importantly, these phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. Our findings indicate that subtelomeric CTCF facilitates telomeric DNA replication by promoting TERRA transcription. Our findings also demonstrate that CTCF-driven TERRA transcription acts in cis to facilitate telomere repeat replication and chromosome stability.},
}

Base Sequence
CCCTC-Binding Factor/*genetics/metabolism
CRISPR-Cas Systems
*DNA Replication
DNA-Binding Proteins/*genetics/metabolism
Gene Editing/methods
HCT116 Cells
Humans
Mutation
Protein Binding
Telomere/*genetics/metabolism
Transcription Factors/*genetics/metabolism
*Transcription, Genetic

@article {pmid29235467,
year = {2017},
author = {Wu, Q and Ferry, QRV and Baeumler, TA and Michaels, YS and Vitsios, DM and Habib, O and Arnold, R and Jiang, X and Maio, S and Steinkraus, BR and Tapia, M and Piazza, P and Xu, N and Holländer, GA and Milne, TA and Kim, JS and Enright, AJ and Bassett, AR and Fulga, TA},
title = {In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {2109},
doi = {10.1038/s41467-017-00686-2},
pmid = {29235467},
issn = {2041-1723},
support = {//Wellcome Trust/United Kingdom ; 340560//European Research Council/International ; G0902418//Medical Research Council/United Kingdom ; MC_UU_12009/6//Medical Research Council/United Kingdom ; 090532/Z/09/Z//Wellcome Trust/United Kingdom ; 105045/Z/14/Z//Wellcome Trust/United Kingdom ; BB/N006550/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {3' Untranslated Regions/genetics ; Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Genome/genetics ; Humans ; MicroRNAs/genetics ; RNA/*genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; Response Elements/genetics ; },
abstract = {RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.},
}

3' Untranslated Regions/genetics
Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/*methods
Genome/genetics
Humans
MicroRNAs/genetics
RNA/*genetics
Regulatory Sequences, Nucleic Acid/*genetics
Response Elements/genetics

@article {pmid29234012,
year = {2017},
author = {Martin-Malpartida, P and Batet, M and Kaczmarska, Z and Freier, R and Gomes, T and Aragón, E and Zou, Y and Wang, Q and Xi, Q and Ruiz, L and Vea, A and Márquez, JA and Massagué, J and Macias, MJ},
title = {Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {2070},
doi = {10.1038/s41467-017-02054-6},
pmid = {29234012},
issn = {2041-1723},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA034610/CA/NCI NIH HHS/United States ; },
mesh = {Allosteric Regulation/genetics ; *Amino Acid Motifs ; Animals ; Binding Sites/genetics ; Bone Morphogenetic Proteins/metabolism ; CRISPR-Cas Systems ; Cell Lineage/genetics ; Crystallography, X-Ray ; Gene Expression Regulation, Developmental ; Goosecoid Protein/*genetics ; Magnetic Resonance Spectroscopy ; Mice ; Mouse Embryonic Stem Cells ; Promoter Regions, Genetic ; Protein Binding ; Smad3 Protein/*chemistry/genetics ; Smad4 Protein/*chemistry/genetics ; Transforming Growth Factor beta/metabolism ; },
abstract = {Smad transcription factors activated by TGF-β or by BMP receptors form trimeric complexes with Smad4 to target specific genes for cell fate regulation. The CAGAC motif has been considered as the main binding element for Smad2/3/4, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. However, chromatin immunoprecipitation analysis in embryonic stem cells showed extensive binding of Smad2/3/4 to GC-rich cis-regulatory elements. Here, we present the structural basis for specific binding of Smad3 and Smad4 to GC-rich motifs in the goosecoid promoter, a nodal-regulated differentiation gene. The structures revealed a 5-bp consensus sequence GGC(GC)|(CG) as the binding site for both TGF-β and BMP-activated Smads and for Smad4. These 5GC motifs are highly represented as clusters in Smad-bound regions genome-wide. Our results provide a basis for understanding the functional adaptability of Smads in different cellular contexts, and their dependence on lineage-determining transcription factors to target specific genes in TGF-β and BMP pathways.},
}

Allosteric Regulation/genetics
*Amino Acid Motifs
Animals
Binding Sites/genetics
Bone Morphogenetic Proteins/metabolism
CRISPR-Cas Systems
Cell Lineage/genetics
Crystallography, X-Ray
Gene Expression Regulation, Developmental
Goosecoid Protein/*genetics
Magnetic Resonance Spectroscopy
Mice
Mouse Embryonic Stem Cells
Promoter Regions, Genetic
Protein Binding
Smad3 Protein/*chemistry/genetics
Smad4 Protein/*chemistry/genetics
Transforming Growth Factor beta/metabolism

@article {pmid29233960,
year = {2017},
author = {Winters, IP and Chiou, SH and Paulk, NK and McFarland, CD and Lalgudi, PV and Ma, RK and Lisowski, L and Connolly, AJ and Petrov, DA and Kay, MA and Winslow, MM},
title = {Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {2053},
doi = {10.1038/s41467-017-01519-y},
pmid = {29233960},
issn = {2041-1723},
support = {R01 HL064274/HL/NHLBI NIH HHS/United States ; F32 HL119059/HL/NHLBI NIH HHS/United States ; R01 CA207133/CA/NCI NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; R35 GM118165/GM/NIGMS NIH HHS/United States ; P30 CA124435/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; Cost-Benefit Analysis ; DNA Mutational Analysis/economics/*methods ; Feasibility Studies ; Female ; Genomics/economics/methods ; High-Throughput Nucleotide Sequencing/methods ; Male ; Mice ; Mutation ; Neoplasms/*genetics/pathology ; Proto-Oncogene Proteins p21(ras)/*genetics ; Recombinational DNA Repair/*genetics ; Reproducibility of Results ; },
abstract = {Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras HDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.},
}

Animals
CRISPR-Cas Systems/genetics
Carcinogenesis/*genetics
Cost-Benefit Analysis
DNA Mutational Analysis/economics/*methods
Feasibility Studies
Female
Genomics/economics/methods
High-Throughput Nucleotide Sequencing/methods
Male
Mice
Mutation
Neoplasms/*genetics/pathology
Proto-Oncogene Proteins p21(ras)/*genetics
Recombinational DNA Repair/*genetics
Reproducibility of Results

@article {pmid29187504,
year = {2018},
author = {DeLay, BD and Corkins, ME and Hanania, HL and Salanga, M and Deng, JM and Sudou, N and Taira, M and Horb, ME and Miller, RK},
title = {Tissue-Specific Gene Inactivation in Xenopus laevis: Knockout of lhx1 in the Kidney with CRISPR/Cas9.},
journal = {Genetics},
volume = {208},
number = {2},
pages = {673-686},
doi = {10.1534/genetics.117.300468},
pmid = {29187504},
issn = {1943-2631},
support = {K01 DK092320/DK/NIDDK NIH HHS/United States ; P40 OD010997/OD/NIH HHS/United States ; R01 HD084409/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; *Gene Silencing ; Gene Targeting ; Kidney/metabolism ; LIM-Homeodomain Proteins/genetics ; Organ Specificity/genetics ; Phenotype ; RNA, Guide ; Transcription Factors/genetics ; Xenopus Proteins/genetics ; Xenopus laevis/*genetics ; },
abstract = {Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis, is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene (slc45a2) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus, providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.},
}

Animals
CRISPR-Cas Systems
Gene Editing
Gene Knockout Techniques
*Gene Silencing
Gene Targeting
Kidney/metabolism
LIM-Homeodomain Proteins/genetics
Organ Specificity/genetics
Phenotype
RNA, Guide
Transcription Factors/genetics
Xenopus Proteins/genetics
Xenopus laevis/*genetics

@article {pmid29167442,
year = {2017},
author = {Lian, J and HamediRad, M and Hu, S and Zhao, H},
title = {Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1688},
doi = {10.1038/s41467-017-01695-x},
pmid = {29167442},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; Cell Membrane/metabolism ; Cellulase/genetics/metabolism ; Gene Deletion ; Genes, Fungal ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; RNA Interference ; Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Transcriptional Activation ; beta Carotene/biosynthesis ; },
abstract = {Designing an optimal microbial cell factory often requires overexpression, knock-down, and knock-out of multiple gene targets. Unfortunately, such rewiring of cellular metabolism is often carried out sequentially and with low throughput. Here, we report a combinatorial metabolic engineering strategy based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae. This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. We demonstrate the application of CRISPR-AID not only to increase the production of β-carotene by 3-fold in a single step, but also to achieve 2.5-fold improvement in the display of an endoglucanase on the yeast surface by optimizing multiple metabolic engineering targets in a combinatorial manner.},
}

*CRISPR-Cas Systems
Cell Membrane/metabolism
Cellulase/genetics/metabolism
Gene Deletion
Genes, Fungal
Metabolic Engineering/*methods
Metabolic Networks and Pathways/genetics
RNA Interference
Saccharomyces cerevisiae/genetics/metabolism
Saccharomyces cerevisiae Proteins/genetics/metabolism
Transcriptional Activation
beta Carotene/biosynthesis

@article {pmid29162626,
year = {2018},
author = {Lansdon, LA and Darbro, BW and Petrin, AL and Hulstrand, AM and Standley, JM and Brouillette, RB and Long, A and Mansilla, MA and Cornell, RA and Murray, JC and Houston, DW and Manak, JR},
title = {Identification of Isthmin 1 as a Novel Clefting and Craniofacial Patterning Gene in Humans.},
journal = {Genetics},
volume = {208},
number = {1},
pages = {283-296},
doi = {10.1534/genetics.117.300535},
pmid = {29162626},
issn = {1943-2631},
support = {R37 DE008559/DE/NIDCR NIH HHS/United States ; T90 DE023520/DE/NIDCR NIH HHS/United States ; R01 GM083999/GM/NIGMS NIH HHS/United States ; R01 DE021071/DE/NIDCR NIH HHS/United States ; T32 GM008629/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Case-Control Studies ; Cleft Lip/*genetics ; Cleft Palate/*genetics ; Comparative Genomic Hybridization ; Craniofacial Abnormalities/embryology/*genetics ; DNA Copy Number Variations ; Gene Deletion ; Haploinsufficiency ; Humans ; Morphogenesis/*genetics ; Organogenesis/*genetics ; Quantitative Trait Loci ; Thrombospondins/*genetics ; },
abstract = {Orofacial clefts are one of the most common birth defects, affecting 1-2 per 1000 births, and have a complex etiology. High-resolution array-based comparative genomic hybridization has increased the ability to detect copy number variants (CNVs) that can be causative for complex diseases such as cleft lip and/or palate. Utilizing this technique on 97 nonsyndromic cleft lip and palate cases and 43 cases with cleft palate only, we identified a heterozygous deletion of Isthmin 1 in one affected case, as well as a deletion in a second case that removes putative 3' regulatory information. Isthmin 1 is a strong candidate for clefting, as it is expressed in orofacial structures derived from the first branchial arch and is also in the same "synexpression group" as fibroblast growth factor 8 and sprouty RTK signaling antagonist 1a and 2, all of which have been associated with clefting. CNVs affecting Isthmin 1 are exceedingly rare in control populations, and Isthmin 1 scores as a likely haploinsufficiency locus. Confirming its role in craniofacial development, knockdown or clustered randomly interspaced short palindromic repeats/Cas9-generated mutation of isthmin 1 in Xenopus laevis resulted in mild to severe craniofacial dysmorphologies, with several individuals presenting with median clefts. Moreover, knockdown of isthmin 1 produced decreased expression of LIM homeobox 8, itself a gene associated with clefting, in regions of the face that pattern the maxilla. Our study demonstrates a successful pipeline from CNV identification of a candidate gene to functional validation in a vertebrate model system, and reveals Isthmin 1 as both a new human clefting locus as well as a key craniofacial patterning gene.},
}

CRISPR-Cas Systems
Case-Control Studies
Cleft Lip/*genetics
Cleft Palate/*genetics
Comparative Genomic Hybridization
Craniofacial Abnormalities/embryology/*genetics
DNA Copy Number Variations
Gene Deletion
Haploinsufficiency
Humans
Morphogenesis/*genetics
Organogenesis/*genetics
Quantitative Trait Loci
Thrombospondins/*genetics

@article {pmid29089570,
year = {2017},
author = {Moder, M and Velimezi, G and Owusu, M and Mazouzi, A and Wiedner, M and Ferreira da Silva, J and Robinson-Garcia, L and Schischlik, F and Slavkovsky, R and Kralovics, R and Schuster, M and Bock, C and Ideker, T and Jackson, SP and Menche, J and Loizou, JI},
title = {Parallel genome-wide screens identify synthetic viable interactions between the BLM helicase complex and Fanconi anemia.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1238},
doi = {10.1038/s41467-017-01439-x},
pmid = {29089570},
issn = {2041-1723},
support = {R01 ES014811/ES/NIEHS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; DNA Damage ; DNA Helicases/genetics ; DNA Repair/*genetics ; Fanconi Anemia/*genetics ; Fanconi Anemia Complementation Group C Protein/*genetics ; Fanconi Anemia Complementation Group D2 Protein/genetics ; Fanconi Anemia Complementation Group Proteins/genetics ; HEK293 Cells ; Haploidy ; Humans ; Mutagenesis, Insertional ; NAD(P)H Dehydrogenase (Quinone)/genetics ; RecQ Helicases/*genetics ; },
abstract = {Maintenance of genome integrity via repair of DNA damage is a key biological process required to suppress diseases, including Fanconi anemia (FA). We generated loss-of-function human haploid cells for FA complementation group C (FANCC), a gene encoding a component of the FA core complex, and used genome-wide CRISPR libraries as well as insertional mutagenesis to identify synthetic viable (genetic suppressor) interactions for FA. Here we show that loss of the BLM helicase complex suppresses FANCC phenotypes and we confirm this interaction in cells deficient for FA complementation group I and D2 (FANCI and FANCD2) that function as part of the FA I-D2 complex, indicating that this interaction is not limited to the FA core complex, hence demonstrating that systematic genome-wide screening approaches can be used to reveal genetic viable interactions for DNA repair defects.},
}

CRISPR-Cas Systems
Cell Line
DNA Damage
DNA Helicases/genetics
DNA Repair/*genetics
Fanconi Anemia/*genetics
Fanconi Anemia Complementation Group C Protein/*genetics
Fanconi Anemia Complementation Group D2 Protein/genetics
Fanconi Anemia Complementation Group Proteins/genetics
HEK293 Cells
Haploidy
Humans
Mutagenesis, Insertional
NAD(P)H Dehydrogenase (Quinone)/genetics
RecQ Helicases/*genetics

@article {pmid27325716,
year = {2015},
author = {},
title = {A Conversation with Jennifer Doudna.},
journal = {Cold Spring Harbor symposia on quantitative biology},
volume = {80},
number = {},
pages = {314-315},
doi = {10.1101/sqb.2015.80.030072},
pmid = {27325716},
issn = {1943-4456},
mesh = {Animals ; Bacteria/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Genetic Engineering ; Genetic Therapy ; Humans ; RNA, Guide/genetics/metabolism ; },
}

Animals
Bacteria/genetics
CRISPR-Cas Systems/*genetics
Gene Editing
Genetic Engineering
Genetic Therapy
Humans
RNA, Guide/genetics/metabolism

@article {pmid27325713,
year = {2015},
author = {},
title = {A Conversation with Emmanuelle Charpentier.},
journal = {Cold Spring Harbor symposia on quantitative biology},
volume = {80},
number = {},
pages = {305-307},
doi = {10.1101/sqb.2015.80.030007},
pmid = {27325713},
issn = {1943-4456},
mesh = {Bacteria/genetics ; CRISPR-Cas Systems/genetics ; Genetic Engineering ; Humans ; RNA/genetics ; Repetitive Sequences, Nucleic Acid/*genetics ; },
}

Bacteria/genetics
CRISPR-Cas Systems/genetics
Genetic Engineering
Humans
RNA/genetics
Repetitive Sequences, Nucleic Acid/*genetics

@article {pmid30266728,
year = {2018},
author = {Morovic, W and Roos, P and Zabel, B and Hidalgo-Cantabrana, C and Kiefer, A and Barrangou, R},
title = {Transcriptional and functional analysis of Bifidobacterium animalis subsp. lactis exposure to tetracycline.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1128/AEM.01999-18},
pmid = {30266728},
issn = {1098-5336},
abstract = {Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019, and employed RNA-seq to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to viable genome copies from droplet digital PCR, and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multi-drug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was up-regulated only during sub-inhibitory tetracycline concentrations, while two novel tetracycline resistance genes were up-regulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were up-regulated while genes for complex carbohydrate utilization, protein metabolism, and CRISPR-Cas systems were down-regulated. These results provide high-throughput means for assessing antibiotic resistance and determine the genetic network that contributes to the global tetracycline response between two highly related probiotic strains.IMPORTANCEBifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.},
}

@article {pmid30261908,
year = {2018},
author = {Dank, A and Smid, EJ and Notebaart, RA},
title = {CRISPR-Cas genome engineering of esterase activity in Saccharomyces cerevisiae steers aroma formation.},
journal = {BMC research notes},
volume = {11},
number = {1},
pages = {682},
doi = {10.1186/s13104-018-3788-5},
pmid = {30261908},
issn = {1756-0500},
abstract = {OBJECTIVE: Saccharomyces cerevisiae is used worldwide for the production of ale-type beers. This yeast is responsible for the production of the characteristic fruity aroma compounds. Esters constitute an important group of aroma active secondary metabolites produced by S. cerevisiae. Previous work suggests that esterase activity, which results in ester degradation, may be the key factor determining the abundance of fruity aroma compounds. Here, we test this hypothesis by deletion of two S. cerevisiae esterases, IAH1 and TIP1, using CRISPR-Cas9 genome editing and by studying the effect of these deletions on esterase activity and extracellular ester pools.

RESULTS: Saccharomyces cerevisiae mutants were constructed lacking esterase IAH1 and/or TIP1 using CRISPR-Cas9 genome editing. Esterase activity using 5-(6)-carboxyfluorescein diacetate (cFDA) as substrate was found to be significantly lower for ΔIAH1 and ΔIAH1ΔTIP1 mutants compared to wild type (WT) activity (P < 0.05 and P < 0.001, respectively). As expected, we observed an increase in relative abundance of acetate and ethyl esters and an increase in ethyl esters in ΔIAH1 and ΔTIP1, respectively. Interestingly, the double gene disruption mutant ΔIAH1ΔTIP1 showed an aroma profile comparable to WT levels, suggesting the existence and activation of a complex regulatory mechanism to compensate multiple genomic alterations in aroma metabolism.},
}

@article {pmid30261168,
year = {2018},
author = {Hidalgo-Cantabrana, C and Goh, YJ and Barrangou, R},
title = {Characterization and Repurposing of Type I and Type II CRISPR-Cas Systems in Bacteria.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jmb.2018.09.013},
pmid = {30261168},
issn = {1089-8638},
abstract = {CRISPR-Cas systems constitute the adaptive immune system of bacteria and archaea, as a sequence-specific nucleic acid targeting defense mechanism. The sequence-specific recognition and cleavage of Cas effector complexes has been harnessed to developed CRISPR-based technologies and drive the genome editing revolution underway, due to their efficacy, efficiency and ease of implementation in a broad range of organisms. CRISPR-based technologies offer a wide variety of opportunities in genome remodeling and transcriptional regulation, opening new avenues for therapeutic and biotechnological applications. To repurpose CRISPR-Cas systems for these applications, the various elements of the system need to be first identified and functionally characterized in their native host. Bioinformatic tools are first used to identify putative CRISPR arrays and their associated genes, followed by a comprehensive characterization of the CRISPR-Cas system, encompassing predictions for guide and target sequences. Subsequently, interference assays and transcriptomic analyses should be performed to probe the functionality of the CRISPR-Cas system. Once an endogenous CRISPR-Cas system is characterized as functional, they can be readily repurposed by delivering an engineered synthetic CRISPR array or a small RNA guide for targeted gene manipulation. Alternatively, developing a plasmid-based system for heterologous expression of the necessary CRISPR components can enable exploitation in other organisms. Altogether, there is a wide diversity of native CRISPR-Cas systems in many bacteria and most archaea that await functional characterization and repurposing for genome editing applications in prokaryotes.},
}

@article {pmid30260068,
year = {2018},
author = {Ebrahimi, S and Teimoori, A and Khanbabaei, H and Tabasi, M},
title = {Harnessing CRISPR/Cas 9 System for manipulation of DNA virus genome.},
journal = {Reviews in medical virology},
volume = {},
number = {},
pages = {e2009},
doi = {10.1002/rmv.2009},
pmid = {30260068},
issn = {1099-1654},
abstract = {The recent development of the Clustered Regularly Interspaced Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a genome editing system, has many potential applications in virology. The possibility of introducing site specific breaks has provided new possibilities to precisely manipulate viral genomics. Here, we provide diagrams to summarize the steps involved in the process. We also systematically review recent applications of the CRISPR/Cas9 system for manipulation of DNA virus genomics and discuss the therapeutic potential of the system to treat viral diseases.},
}

@article {pmid30258941,
year = {2018},
author = {Karlapudi, AP and T C, V and Tammineedi, J and Srirama, K and Kanumuri, L and Prabhakar Kodali, V},
title = {In silico sgRNA tool design for CRISPR control of quorum sensing in Acinetobacter species.},
journal = {Genes & diseases},
volume = {5},
number = {2},
pages = {123-129},
doi = {10.1016/j.gendis.2018.03.004},
pmid = {30258941},
issn = {2352-3042},
abstract = {CRISPR genome editing utilizes Cas9 nuclease and single guide RNA (sgRNA), which directs the nuclease to a specific site in the genome and makes a double-stranded break (DSB). Design of sgRNA for CRISPR-Cas targeting, and to promote CRISPR adaptation, uses a regulatory mechanism that ensures maximum CRISPR-Cas9 system functions when a bacterial population is at highest risk of phage infection. Acinetobacter baumannii is the most regularly identified gram-negative bacterium infecting patients. Recent reports have demonstrated that the extent of diseases caused by A. baumannii is expanding and, in a few cases, now surpasses the quantity of infections caused by P. aeruginosa. Most Acinetobacter strains possess biofilm-forming ability, which plays a major role in virulence and drug resistance. Biofilm bacteria use quorum sensing, a cell-to-cell communication process, to activate gene expression. Many genes are involved in biofilm formation and the mechanism to disrupt the biofilm network is still not clearly understood. In this study, we performed in silico gene editing to exploit the AbaI gene, responsible for biofilm formation. The study explored different tools available for genome editing to create gene knockouts, selecting the A. baumannii AbaI gene as a target.},
}

@article {pmid29621903,
year = {2018},
author = {Shao, J and Glorieux, C and Liao, J and Chen, P and Lu, W and Liang, Z and Wen, S and Hu, Y and Huang, P},
title = {Impact of Nrf2 on tumour growth and drug sensitivity in oncogenic K-ras-transformed cells in vitro and in vivo.},
journal = {Free radical research},
volume = {52},
number = {6},
pages = {661-671},
doi = {10.1080/10715762.2018.1462494},
pmid = {29621903},
issn = {1029-2470},
mesh = {Animals ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Transformation, Neoplastic/*genetics/metabolism/pathology ; Doxorubicin/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Female ; Gene Editing/methods ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; HEK293 Cells ; Heme Oxygenase-1/genetics/metabolism ; Humans ; Isothiocyanates/pharmacology ; Membrane Proteins/genetics/metabolism ; Mice ; Mice, Nude ; NAD(P)H Dehydrogenase (Quinone)/genetics/metabolism ; NF-E2-Related Factor 2/deficiency/*genetics ; Proto-Oncogene Proteins p21(ras)/*genetics/metabolism ; Reactive Oxygen Species/metabolism ; Signal Transduction ; Tumor Burden/drug effects ; },
abstract = {K-ras is one of the most common oncogenes in human cancers, and its aberrant activation may lead to malignant transformation associated with oxidative stress and activation of the transcription factor Nrf2 that regulates multiple detoxification enzymes. The purpose of this research was to use gene editing technology to evaluate the role of Nrf2 in affecting tumour growth and drug sensitivity of K-rasG12V-transformed cells. We showed that induction of K-rasG12V caused a significant activation of Nrf2 associated with increased expression of its target genes NAD(P)H:quinone oxidoreductase 1 (NQO1) and haem oxygenase-1 (HO-1). Interestingly, knock-out of Nrf2 by CRISPR/Cas9 in K-rasG12V-expressing cells only impacted the expression of NQO1 but not HO-1. We also found that Nrf2 knock-out caused high reactive oxygen species (ROS) stress, suppression of cell proliferation, increased apoptosis in vitro, and a decrease of tumour growth in vivo. Furthermore, abrogation of Nrf2 significantly increased the sensitivity of K-rasG12V cells to multiple anticancer agents including phenethyl isothiocyanate (PEITC), doxorubicin, etoposide, and cisplatin. These results show that genetic abrogation of Nrf2 impairs the malignant phenotype of K-RasG12V-transformed cells in vitro and in vivo, and demonstrates the critical role of Nrf2 in promoting cell survival and drug resistance in cells harbouring oncogenic K-ras. As such, inhibition of Nrf2 would be an attractive strategy to increase the therapeutic effect and overcome drug resistance in cancer with oncogenic K-ras activation.},
}

Animals
Antineoplastic Agents/pharmacology
CRISPR-Cas Systems
Cell Line, Tumor
Cell Proliferation/drug effects
Cell Transformation, Neoplastic/*genetics/metabolism/pathology
Doxorubicin/pharmacology
Drug Resistance, Neoplasm/drug effects/*genetics
Female
Gene Editing/methods
*Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
HEK293 Cells
Heme Oxygenase-1/genetics/metabolism
Humans
Isothiocyanates/pharmacology
Membrane Proteins/genetics/metabolism
Mice
Mice, Nude
NAD(P)H Dehydrogenase (Quinone)/genetics/metabolism
NF-E2-Related Factor 2/deficiency/*genetics
Proto-Oncogene Proteins p21(ras)/*genetics/metabolism
Reactive Oxygen Species/metabolism
Signal Transduction
Tumor Burden/drug effects

@article {pmid29391173,
year = {2018},
author = {Inoue, YU and Morimoto, Y and Hoshino, M and Inoue, T},
title = {Generation of Pax6-IRES-EGFP knock-in mouse via the cloning-free CRISPR/Cas9 system to reliably visualize neurodevelopmental dynamics.},
journal = {Neuroscience research},
volume = {132},
number = {},
pages = {1-7},
doi = {10.1016/j.neures.2018.01.007},
pmid = {29391173},
issn = {1872-8111},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; *Gene Knock-In Techniques ; Genes, Reporter/genetics ; Genes, erbB-1/*genetics ; Mice, Knockout ; PAX6 Transcription Factor/*genetics ; RNA, Guide/genetics ; },
abstract = {Pax6 encodes a transcription factor that plays pivotal roles in eye development, early brain patterning, neocortical arealization, and so forth. Visualization of Pax6 expression dynamics in these events could offer numerous advantages to neurodevelopmental studies. While CRISPR/Cas9 system has dramatically accelerated one-step generation of knock-out mouse, establishment of gene-cassette knock-in mouse via zygote injection has been considered insufficient due to its low efficiency. Recently, an improved CRISPR/Cas9 system for effective gene-cassette knock-in has been reported, where the native form of guide RNAs (crRNA and tracrRNA) assembled with recombinant Cas9 protein are directly delivered into mouse fertilized eggs. Here we apply this strategy to insert IRES-EGFP-pA cassette into Pax6 locus and achieve efficient targeted insertions of the 1.8 kb reporter gene. In Pax6-IRES-EGFP mouse we have generated, EGFP-positive cells reside in the eyes and cerebellum as endogenous Pax6 expressing cells at postnatal day 2. At the early embryonic stages when the embryos are transparent, EGFP-positive regions can be easily identified without PCR-based genotyping, precisely recapitulating the endogenous Pax6 expression patterns. Remarkably, at E12.5, the graded expression patterns of Pax6 in the developing neocortex now become recognizable in our knock-in mice, serving a sufficiently sensitive and useful tool to precisely visualize neurodevelopmental processes.},
}

Animals
CRISPR-Cas Systems/*genetics
*Gene Knock-In Techniques
Genes, Reporter/genetics
Genes, erbB-1/*genetics
Mice, Knockout
PAX6 Transcription Factor/*genetics
RNA, Guide/genetics

@article {pmid29285717,
year = {2018},
author = {Puchta, H},
title = {Broadening the applicability of CRISPR/Cas9 in plants.},
journal = {Science China. Life sciences},
volume = {61},
number = {1},
pages = {126-127},
doi = {10.1007/s11427-017-9249-3},
pmid = {29285717},
issn = {1869-1889},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*trends ; Plants/*genetics ; Plants, Genetically Modified/genetics ; },
}

CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing/*trends
Plants/*genetics
Plants, Genetically Modified/genetics

@article {pmid29285711,
year = {2018},
author = {Meng, X and Hu, X and Liu, Q and Song, X and Gao, C and Li, J and Wang, K},
title = {Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice.},
journal = {Science China. Life sciences},
volume = {61},
number = {1},
pages = {122-125},
doi = {10.1007/s11427-017-9247-9},
pmid = {29285711},
issn = {1869-1889},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Genes, Plant/genetics ; Genome, Plant/*genetics ; Mutation ; Oryza/*genetics ; },
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/*methods
Genes, Plant/genetics
Genome, Plant/*genetics
Mutation
Oryza/*genetics

@article {pmid29247562,
year = {2018},
author = {Alexander, WG},
title = {A history of genome editing in Saccharomyces cerevisiae.},
journal = {Yeast (Chichester, England)},
volume = {35},
number = {5},
pages = {355-360},
doi = {10.1002/yea.3300},
pmid = {29247562},
issn = {1097-0061},
mesh = {CRISPR-Cas Systems ; Gene Editing/*history ; *Genome, Fungal ; History, 20th Century ; History, 21st Century ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Genome editing is a form of highly precise genetic engineering which produces alterations to an organism's genome as small as a single base pair with no incidental or auxiliary modifications; this technique is crucial to the field of synthetic biology, which requires such precision in the installation of novel genetic circuits into host genomes. While a new methodology for most organisms, genome editing capabilities have been used in the budding yeast Saccharomyces cerevisiae for decades. In this review, I will present a brief history of genome editing in S. cerevisiae, discuss the current gold standard method of Cas9-mediated genome editing, and speculate on future directions of the field.},
}

CRISPR-Cas Systems
Gene Editing/*history
*Genome, Fungal
History, 20th Century
History, 21st Century
Saccharomyces cerevisiae/*genetics

@article {pmid29192328,
year = {2018},
author = {Sun, L and Kao, TH},
title = {CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.},
journal = {Plant reproduction},
volume = {31},
number = {2},
pages = {129-143},
doi = {10.1007/s00497-017-0314-1},
pmid = {29192328},
issn = {2194-7961},
support = {IOS-1645557//National Science Foundation/International ; },
mesh = {Alleles ; *CRISPR-Cas Systems ; F-Box Proteins/genetics/*metabolism ; Flowers/enzymology/genetics/physiology ; Gene Knockout Techniques ; Petunia/enzymology/*genetics/physiology ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Pollen/enzymology/genetics/physiology ; Pollination ; Proteasome Endopeptidase Complex/genetics/*metabolism ; Ribonucleases/*genetics/metabolism ; Self-Incompatibility in Flowering Plants/*genetics ; },
abstract = {KEY MESSAGE: Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2-haplotype and S 3-haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S 3-RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.},
}

Alleles
*CRISPR-Cas Systems
F-Box Proteins/genetics/*metabolism
Flowers/enzymology/genetics/physiology
Gene Knockout Techniques
Petunia/enzymology/*genetics/physiology
Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Pollen/enzymology/genetics/physiology
Pollination
Proteasome Endopeptidase Complex/genetics/*metabolism
Ribonucleases/*genetics/metabolism
Self-Incompatibility in Flowering Plants/*genetics

@article {pmid30255296,
year = {2018},
author = {Adames, NR and Gallegos, JE and Peccoud, J},
title = {Yeast genetic interaction screens in the age of CRISPR/Cas.},
journal = {Current genetics},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00294-018-0887-8},
pmid = {30255296},
issn = {1432-0983},
support = {R01GM078989//National Institute of General Medical Sciences/ ; 1759900//Directorate for Biological Sciences/ ; 1832320//Directorate for Engineering/ ; },
abstract = {The ease of performing both forward and reverse genetics in Saccharomyces cerevisiae, along with its stable haploid state and short generation times, has made this budding yeast the consummate model eukaryote for genetics. The major advantage of using budding yeast for reverse genetics is this organism's highly efficient homology-directed repair, allowing for precise genome editing simply by introducing DNA with homology to the chromosomal target. Although plasmid- and PCR-based genome editing tools are quite efficient, they depend on rare spontaneous DNA breaks near the target sequence. Consequently, they can generate only one genomic edit at a time, and the edit must be associated with a selectable marker. However, CRISPR/Cas technology is efficient enough to permit markerless and multiplexed edits in a single step. These features have made CRISPR/Cas popular for yeast strain engineering in synthetic biology and metabolic engineering applications, but it has not been widely employed for genetic screens. In this review, we critically examine different methods to generate multi-mutant strains in systematic genetic interaction screens and discuss the potential of CRISPR/Cas to supplement or improve on these methods.},
}

@article {pmid30251378,
year = {2018},
author = {da Silva Xavier, A and de Almeida, JCF and de Melo, AG and Rousseau, GM and Tremblay, DM and de Rezende, RR and Moineau, S and Alfenas-Zerbini, P},
title = {Characterization of CRISPR-Cas systems in the Ralstonia solanacearum Species Complex.},
journal = {Molecular plant pathology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mpp.12750},
pmid = {30251378},
issn = {1364-3703},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPRs) is an array of short DNA repeat sequences separated by unique spacer sequences that is flanked by associated (Cas) genes. CRISPR-Cas systems are found in the genome of several microbes and they can act as an adaptive immune mechanism against invading foreign nucleic acids such as phage genomes. Here, we studied the CRISPR-Cas systems in plant pathogenic bacteria of the Ralstonia solanacearum species complex (RSSC). A CRISPR-Cas system was found in 31% of RSSC genomes present in public databases. Specifically, CRISPR-Cas types I-E and II-C were found, with I-E being the most common. The presence of the same CRISPR-Cas types in distinct Ralstonia phylotypes and species suggests the acquisition of the system by a common ancestor before Ralstonia species segregation. Additionally, a Cas1 phylogeny (I-E type) showed a perfect geographical segregation of phylotypes, supporting an ancient acquisition. Ralstonia solanacearum strains CFBP2957 and K60T were challenged with a virulent phage, and the CRISPR arrays of bacteriophage-insensitive mutants (BIMs) were analyzed. No new spacer acquisition was detected in the analyzed BIMs. The functionality of the CRISPR-Cas interference step was also tested in R. solanacearum CFBP2957 using a spacer-PAM delivery system and no resistance was observed against phage phiAP1. Our results showed that the CRISPR-Cas system in R. solanacearum CFBP2957 is not its primary antiviral strategy. This article is protected by copyright. All rights reserved.},
}

@article {pmid30251319,
year = {2018},
author = {Martin, RM and Moniruzzaman, M and Mucci, NC and Willis, A and Woodhouse, JN and Xian, Y and Xiao, C and Brussaard, CPD and Wilhelm, SW},
title = {Cylindrospermopsis raciborskii Virus and host: Genomic characterization and ecological relevance.},
journal = {Environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1462-2920.14425},
pmid = {30251319},
issn = {1462-2920},
abstract = {Cylindrospermopsis (Raphidiopsis) raciborskii is an invasive, filamentous, nitrogen-fixing cyanobacterium that forms frequent blooms in freshwater habitats. While viruses play key roles in regulating the abundance, production, and diversity of their hosts in aquatic ecosystems, the role(s) of viruses in the ecology of C. raciborskii is almost unexplored. Progress in this field has been hindered by the absence of a characterized virus-host system in C. raciborskii. To bridge this gap, we sequenced the genome of CrV-01T, a previously isolated cyanosiphovirus, and its host C. raciborskii strain Cr2010. Analyses suggest that CrV-01T represents a distinct clade of siphoviruses infecting, and perhaps lysogenizing, filamentous cyanobacteria. Its genome contains unique features that include an intact CRISPR array and a 12-kb inverted duplication. Evidence suggests CrV-01T recently gained the ability to infect Cr2010 and recently lost the ability to form lysogens. The cyanobacterial host contains a CRISPR-Cas system with CRISPR spacers matching protospacers within the inverted duplication of the CrV-01T genome. Examination of metagenomes demonstrates that viruses with high genetic identity to CrV-01T, but lacking the inverted duplication, are present in C. raciborskii blooms in Australia. The unique genomic features of the CrV/Cr2010 system offers opportunities to investigate in more detail virus-host interactions in an ecologically important bloom-forming cyanobacterium. This article is protected by copyright. All rights reserved.},
}

@article {pmid30002084,
year = {2018},
author = {Shayevitch, R and Askayo, D and Keydar, I and Ast, G},
title = {The importance of DNA methylation of exons on alternative splicing.},
journal = {RNA (New York, N.Y.)},
volume = {24},
number = {10},
pages = {1351-1362},
doi = {10.1261/rna.064865.117},
pmid = {30002084},
issn = {1469-9001},
mesh = {*Alternative Splicing ; CRISPR-Cas Systems ; Cell Line ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; *DNA Methylation ; *Exons ; Gene Editing ; Gene Targeting ; Humans ; Introns ; RNA, Guide ; },
abstract = {Alternative splicing (AS) contributes to proteome diversity. As splicing occurs cotranscriptionally, epigenetic determinants such as DNA methylation likely play a part in regulation of AS. Previously, we have shown that DNA methylation marks exons and that a loss of DNA methylation alters splicing patterns in a genome-wide manner. To investigate the influence of DNA methylation on splicing of individual genes, we developed a method to manipulate DNA methylation in vivo in a site-specific manner using the deactivated endonuclease Cas9 fused to enzymes that methylate or demethylate DNA. We used this system to directly change the DNA methylation pattern of selected exons and introns. We demonstrated that changes in the methylation pattern of alternatively spliced exons, but not constitutively spliced exons or introns, altered inclusion levels. This is the first direct demonstration that DNA methylation of exon-encoding regions is directly involved in regulation of AS.},
}

*Alternative Splicing
CRISPR-Cas Systems
Cell Line
DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism
*DNA Methylation
*Exons
Gene Editing
Gene Targeting
Humans
Introns
RNA, Guide

@article {pmid29849821,
year = {2018},
author = {Huang, LC and Tam, KW and Liu, WN and Lin, CY and Hsu, KW and Hsieh, WS and Chi, WM and Lee, AW and Yang, JM and Lin, CL and Lee, CH},
title = {CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer.},
journal = {Disease markers},
volume = {2018},
number = {},
pages = {3835783},
doi = {10.1155/2018/3835783},
pmid = {29849821},
issn = {1875-8630},
mesh = {Antineoplastic Agents/pharmacology ; *CRISPR-Cas Systems ; Cell Cycle/drug effects ; Cell Death/drug effects ; Cell Line, Tumor ; Gene Editing/*methods ; Humans ; Protein Kinase Inhibitors/pharmacology ; Quinazolines/pharmacology ; Receptor, Epidermal Growth Factor/*genetics ; Thyroid Carcinoma, Anaplastic/*genetics ; Thyroid Neoplasms/*genetics ; },
abstract = {Anaplastic carcinoma of the thyroid (ATC), also called undifferentiated thyroid cancer, is the least common but most aggressive and deadly thyroid gland malignancy of all thyroid cancers. The aim of this study is to explore essential biomarker and use CRISPR/Cas9 with lentivirus delivery to establish a gene-target therapeutic platform in ATC cells. At the beginning, the gene expression datasets from 1036 cancers from CCLE and 8215 tumors from TCGA were collected and analyzed, showing EGFR is predominantly overexpressed in thyroid cancers than other type of cancers (P = 0.017 in CCLE and P = 0.001 in TCGA). Using CRISPR/Cas9 genomic edit system, ATC cells with EGFR sgRNA lentivirus transfection obtained great disruptions on gene and protein expression, resulting in cell cycle arrest, cell growth inhibition, and most importantly metastasis turn-off ability. In addition, the FDA-approved TKI of afatinib for EGFR targeting also illustrates great anticancer activity on cancer cell death occurrence, cell growth inhibition, and cell cycle arrest in SW579 cells, an EGFR expressing human ATC cell line. Furthermore, off-target effect of using EGFR sgRNAs was measured and found no genomic editing can be detected in off-target candidate gene. To conclude, this study provides potential ATC therapeutic strategies for current and future clinical needs, which may be possible in increasing the survival rate of ATC patients by translational medicine.},
}

Antineoplastic Agents/pharmacology
*CRISPR-Cas Systems
Cell Cycle/drug effects
Cell Death/drug effects
Cell Line, Tumor
Gene Editing/*methods
Humans
Protein Kinase Inhibitors/pharmacology
Quinazolines/pharmacology
Receptor, Epidermal Growth Factor/*genetics
Thyroid Carcinoma, Anaplastic/*genetics
Thyroid Neoplasms/*genetics

@article {pmid29732484,
year = {2018},
author = {Franco-Tormo, MJ and Salas-Crisostomo, M and Rocha, NB and Budde, H and Machado, S and Murillo-Rodríguez, E},
title = {CRISPR/Cas9, the Powerful New Genome-Editing Tool for Putative Therapeutics in Obesity.},
journal = {Journal of molecular neuroscience : MN},
volume = {65},
number = {1},
pages = {10-16},
doi = {10.1007/s12031-018-1076-4},
pmid = {29732484},
issn = {1559-1166},
support = {CN-17-19//University of California Institute for Mexico and the United States/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Obesity/*genetics/therapy ; },
abstract = {The molecular technology known as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is revolutionizing the field of medical research and deepening our understanding of numerous biological processes. The attraction of CRISPR/Cas9 lies in its ability to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms, a technology that was once considered either too expensive or scientifically risky. CRISPR/Cas9 has been successfully applied in agriculture to develop the next generation of disease-resistant plants. Now, the capability of gene editing has been translated to the biomedical area, focusing on the future of medicine faced with drug-resistant microbes by selectively targeting genes involved in antibiotic resistance, for example, or finding the ultimate strategy for cancer or HIV. In this regard, it was recently demonstrated that an injection of cancer-fighting CRISPR-modified white blood cells in a patient suffering from metastatic lung cancer could lead to promising results. Researchers and bioethicists are debating questions about the regulation of CRISPR/Cas9 that must be addressed. While legal challenges surround the use of this technique for genetically modifying cell lines in humans, we review the basic understanding of CRISPR/Cas9 and discuss how this technology could represent a candidate for treatment of non-communicable diseases in nutrition, such as obesity.},
}

Animals
*CRISPR-Cas Systems
Gene Editing/*methods
Genetic Therapy/*methods
Humans
Obesity/*genetics/therapy