@article {pmid30241607,
year = {2018},
author = {Zhang, C and Konermann, S and Brideau, NJ and Lotfy, P and Wu, X and Novick, SJ and Strutzenberg, T and Griffin, PR and Hsu, PD and Lyumkis, D},
title = {Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d.},
journal = {Cell},
volume = {175},
number = {1},
pages = {212-223.e17},
doi = {10.1016/j.cell.2018.09.001},
pmid = {30241607},
issn = {1097-4172},
abstract = {CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.},
}

@article {pmid30239876,
year = {2018},
author = {Han, W and Stella, S and Zhang, Y and Guo, T and Sulek, K and Peng-Lundgren, L and Montoya, G and She, Q},
title = {A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gky844},
pmid = {30239876},
issn = {1362-4962},
abstract = {Recently, Type III-A CRISPR-Cas systems were found to catalyze the synthesis of cyclic oligoadenylates (cOAs), a second messenger that specifically activates Csm6, a Cas accessory RNase and confers antiviral defense in bacteria. To test if III-B CRISPR-Cas systems could mediate a similar CRISPR signaling pathway, the Sulfolobus islandicus Cmr-α ribonucleoprotein complex (Cmr-α-RNP) was purified from the native host and tested for cOA synthesis. We found that the system showed a robust production of cyclic tetra-adenylate (c-A4), and that c-A4 functions as a second messenger to activate the III-B-associated RNase Csx1 by binding to its CRISPR-associated Rossmann Fold domain. Investigation of the kinetics of cOA synthesis revealed that Cmr-α-RNP displayed positively cooperative binding to the adenosine triphosphate (ATP) substrate. Furthermore, mutagenesis of conserved domains in Cmr2α confirmed that, while Palm 2 hosts the active site of cOA synthesis, Palm 1 domain serves as the primary site in the enzyme-substrate interaction. Together, our data suggest that the two Palm domains cooperatively interact with ATP molecules to achieve a robust cOA synthesis by the III-B CRISPR-Cas system.},
}

@article {pmid30239713,
year = {2018},
author = {Haverkamp, THA and Geslin, C and Lossouarn, J and Podosokorskaya, OA and Kublanov, I and Nesbø, CL},
title = {Thermosipho spp. immune system differences affect variation in genome size and geographical distributions.},
journal = {Genome biology and evolution},
volume = {},
number = {},
pages = {},
doi = {10.1093/gbe/evy202},
pmid = {30239713},
issn = {1759-6653},
abstract = {Thermosipho species inhabit thermal environments such as marine hydrothermal vents, petroleum reservoirs and terrestrial hot springs. A 16S rRNA phylogeny of available Thermosipho spp. sequences suggested habitat specialists adapted to living in hydrothermal vents only, and habitat generalists inhabiting oil reservoirs, hydrothermal vents and hotsprings. Comparative genomics of 15 Thermosipho genomes separated them into three distinct species with different habitat distributions: the widely distributed T. africanus and the more specialized, T. melanesiensis and T. affectus. Moreover, the species can be differentiated on the basis of genome size, genome content and immune system composition. For instance, the T. africanus genomes are largest and contained the most carbohydrate metabolism genes, which could explain why these isolates were obtained from ecologically more divergent habitats. Nonetheless, all the Thermosipho genomes, like other Thermotogae genomes, show evidence of genome streamlining. Genome size differences between the species could further be correlated to differences in defense capacities against foreign DNA, which influence recombination via HGT. The smallest genomes are found in T. affectus that contain both CRISPR-cas Type I and III systems, but no RM system genes. We suggest that this has caused these genomes to be almost devoid of mobile elements, contrasting the two other species genomes that contain a higher abundance of mobile elements combined with different immune system configurations. Taken together, the comparative genomic analyses of Thermosipho spp. revealed genetic variation allowing habitat differentiation within the genus as well as differentiation with respect to invading mobile DNA.},
}

@article {pmid30238143,
year = {2018},
author = {Shi, TQ and Huang, H and Kerkhoven, EJ and Ji, XJ},
title = {Advancing metabolic engineering of Yarrowia lipolytica using the CRISPR/Cas system.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00253-018-9366-x},
pmid = {30238143},
issn = {1432-0614},
support = {21776131//National Natural Science Foundation of China/ ; 21476111//National Natural Science Foundation of China/ ; XTD1814//the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture/ ; },
abstract = {The oleaginous yeast Yarrowia lipolytica is widely used for the production of both bulk and fine chemicals, including organic acids, fatty acid-derived biofuels and chemicals, polyunsaturated fatty acids, single-cell proteins, terpenoids, and other valuable products. Consequently, it is becoming increasingly popular for metabolic engineering applications. Multiple gene manipulation tools including URA blast, Cre/LoxP, and transcription activator-like effector nucleases (TALENs) have been developed for metabolic engineering in Y. lipolytica. However, the low efficiency and time-consuming procedures involved in these methods hamper further research. The emergence of the CRISPR/Cas system offers a potential solution for these problems due to its high efficiency, ease of operation, and time savings, which can significantly accelerate the genomic engineering of Y. lipolytica. In this review, we summarize the research progress on the development of CRISPR/Cas systems for Y. lipolytica, including Cas9 proteins and sgRNA expression strategies, as well as gene knock-out/knock-in and repression/activation applications. Finally, the most promising and tantalizing future prospects in this area are highlighted.},
}

@article {pmid28474250,
year = {2017},
author = {Wang, W and Wang, X},
title = {Single-cell CRISPR screening in drug resistance.},
journal = {Cell biology and toxicology},
volume = {33},
number = {3},
pages = {207-210},
doi = {10.1007/s10565-017-9396-7},
pmid = {28474250},
issn = {1573-6822},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Drug Discovery/methods ; Drug Evaluation, Preclinical/methods ; Drug Resistance, Neoplasm/*genetics ; Humans ; RNA, Guide/genetics ; },
}

Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Drug Discovery/methods
Drug Evaluation, Preclinical/methods
Drug Resistance, Neoplasm/*genetics
Humans
RNA, Guide/genetics

@article {pmid30232153,
year = {2018},
author = {Lander, N and Chiurillo, MA and Bertolini, MS and Storey, M and Vercesi, AE and Docampo, R},
title = {Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of Trypanosoma cruzi.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA118.004498},
pmid = {30232153},
issn = {1083-351X},
abstract = {In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation-dephosphorylation cycle for the E1α subunit of PDH from non-vertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and Trypanosoma brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP:ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting culture host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity and energy metabolism in T. cruzi. Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.},
}

@article {pmid30037991,
year = {2018},
author = {Huang, J and Li, J and Zhou, J and Wang, L and Yang, S and Hurst, LD and Li, WH and Tian, D},
title = {Identifying a large number of high-yield genes in rice by pedigree analysis, whole-genome sequencing, and CRISPR-Cas9 gene knockout.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {32},
pages = {E7559-E7567},
doi = {10.1073/pnas.1806110115},
pmid = {30037991},
issn = {1091-6490},
mesh = {Agriculture/*methods ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques/methods ; Genome, Plant/*physiology ; Oryza/genetics/*physiology ; Pedigree ; Phenotype ; Plants, Genetically Modified/genetics/physiology ; *Quantitative Trait Loci ; *Quantitative Trait, Heritable ; Selection, Genetic/genetics ; Sequence Analysis, DNA/methods ; },
abstract = {Repeated artificial selection of a complex trait facilitates the identification of genes underlying the trait, especially if multiple selected descendant lines are available. Here we developed a pedigree-based approach to identify genes underlying the Green Revolution (GR) phenotype. From a pedigree analysis, we selected 30 cultivars including the "miracle rice" IR8, a GR landmark, its ancestors and descendants, and also other related cultivars for identifying high-yield genes. Through sequencing of these genomes, we identified 28 ancestral chromosomal blocks that were maintained in all the high-yield cultivars under study. In these blocks, we identified six genes of known function, including the GR gene sd1, and 123 loci with genes of unknown function. We randomly selected 57 genes from the 123 loci for knockout or knockdown studies and found that a high proportion of these genes are essential or have phenotypic effects related to rice production. Notably, knockout lines have significant changes in plant height (P < 0.003), a key GR trait, compared with wild-type lines. Some gene knockouts or knockdowns were especially interesting. For example, knockout of Os10g0555100, a putative glucosyltransferase gene, showed both reduced growth and altered panicle architecture. In addition, we found that in some retained chromosome blocks several GR-related genes were clustered, although they have unrelated sequences, suggesting clustering of genes with similar functions. In conclusion, we have identified many high-yield genes in rice. Our method provides a powerful means to identify genes associated with a specific trait.},
}

Agriculture/*methods
CRISPR-Cas Systems/genetics
Gene Knockout Techniques/methods
Genome, Plant/*physiology
Oryza/genetics/*physiology
Pedigree
Phenotype
Plants, Genetically Modified/genetics/physiology
*Quantitative Trait Loci
*Quantitative Trait, Heritable
Selection, Genetic/genetics
Sequence Analysis, DNA/methods

@article {pmid29987021,
year = {2018},
author = {Banerjee, S and Ji, C and Mayfield, JE and Goel, A and Xiao, J and Dixon, JE and Guo, X},
title = {Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {32},
pages = {8155-8160},
doi = {10.1073/pnas.1806797115},
pmid = {29987021},
issn = {1091-6490},
support = {T32 CA009523/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Proliferation/drug effects ; Crystallography, X-Ray ; Curcumin/*pharmacology/therapeutic use ; Drug Synergism ; Female ; Gene Editing/methods ; Gene Knockout Techniques/methods ; HEK293 Cells ; Humans ; Inhibitory Concentration 50 ; Mice ; Neoplasms/*drug therapy/pathology ; Oligopeptides/pharmacology ; Proteasome Endopeptidase Complex/*metabolism ; Proteasome Inhibitors/pharmacology ; Protein-Serine-Threonine Kinases/*antagonists & inhibitors/chemistry/genetics/metabolism ; Protein-Tyrosine Kinases/*antagonists & inhibitors/chemistry/genetics/metabolism ; Signal Transduction/drug effects ; Tumor Burden/drug effects ; Xenograft Model Antitumor Assays ; },
abstract = {Curcumin, the active ingredient in Curcuma longa, has been in medicinal use since ancient times. However, the therapeutic targets and signaling cascades modulated by curcumin have been enigmatic despite extensive research. Here we identify dual-specificity tyrosine-regulated kinase 2 (DYRK2), a positive regulator of the 26S proteasome, as a direct target of curcumin. Curcumin occupies the ATP-binding pocket of DYRK2 in the cocrystal structure, and it potently and specifically inhibits DYRK2 over 139 other kinases tested in vitro. As a result, curcumin diminishes DYRK2-mediated 26S proteasome phosphorylation in cells, leading to reduced proteasome activity and impaired cell proliferation. Interestingly, curcumin synergizes with the therapeutic proteasome inhibitor carfilzomib to induce apoptosis in a variety of proteasome-addicted cancer cells, while this drug combination exhibits modest to no cytotoxicity to noncancerous cells. In a breast cancer xenograft model, curcumin treatment significantly reduces tumor burden in immunocompromised mice, showing a similar antitumor effect as CRISPR/Cas9-mediated DYRK2 depletion. These results reveal an unexpected role of curcumin in DYRK2-proteasome inhibition and provide a proof-of-concept that pharmacological manipulation of proteasome regulators may offer new opportunities for anticancer treatment.},
}

Animals
Antineoplastic Agents/*pharmacology/therapeutic use
Apoptosis/drug effects
CRISPR-Cas Systems
Cell Proliferation/drug effects
Crystallography, X-Ray
Curcumin/*pharmacology/therapeutic use
Drug Synergism
Female
Gene Editing/methods
Gene Knockout Techniques/methods
HEK293 Cells
Humans
Inhibitory Concentration 50
Mice
Neoplasms/*drug therapy/pathology
Oligopeptides/pharmacology
Proteasome Endopeptidase Complex/*metabolism
Proteasome Inhibitors/pharmacology
Protein-Serine-Threonine Kinases/*antagonists & inhibitors/chemistry/genetics/metabolism
Protein-Tyrosine Kinases/*antagonists & inhibitors/chemistry/genetics/metabolism
Signal Transduction/drug effects
Tumor Burden/drug effects
Xenograft Model Antitumor Assays

@article {pmid29669988,
year = {2018},
author = {Ishino, T and Hashimoto, M and Amagasa, M and Saito, N and Dochi, O and Kirisawa, R and Kitamura, H},
title = {Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation.},
journal = {Biomedical research (Tokyo, Japan)},
volume = {39},
number = {2},
pages = {95-104},
doi = {10.2220/biomedres.39.95},
pmid = {29669988},
issn = {1880-313X},
mesh = {Animals ; CRISPR-Cas Systems ; Cattle ; Clone Cells ; Fibroblasts/*metabolism ; *Gene Editing/methods ; Genetic Loci ; Genotype ; HeLa Cells ; Humans ; Mutation ; *Nuclear Transfer Techniques ; Receptor-Like Protein Tyrosine Phosphatases, Class 2/genetics ; Reproducibility of Results ; Transfection ; },
abstract = {Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.},
}

Animals
CRISPR-Cas Systems
Cattle
Clone Cells
Fibroblasts/*metabolism
*Gene Editing/methods
Genetic Loci
Genotype
HeLa Cells
Humans
Mutation
*Nuclear Transfer Techniques
Receptor-Like Protein Tyrosine Phosphatases, Class 2/genetics
Reproducibility of Results
Transfection

@article {pmid29556895,
year = {2018},
author = {Su, X and Cui, K and Du, S and Li, H and Lu, F and Shi, D and Liu, Q},
title = {Efficient genome editing in cultured cells and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system.},
journal = {In vitro cellular & developmental biology. Animal},
volume = {54},
number = {5},
pages = {375-383},
doi = {10.1007/s11626-018-0236-8},
pmid = {29556895},
issn = {1543-706X},
support = {2013AA102504//Chinese High technique project/ ; 31760648//National Natural Science Foundation/ ; 31260552//Natural Science Foundation of Guangxi Province (CN)/ ; },
mesh = {Animals ; *Animals, Genetically Modified ; Buffaloes/genetics ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Fertilization in Vitro ; Fibroblasts ; Gene Editing/*methods ; Mutation ; Myostatin/deficiency/*genetics ; Phenotype ; Swine/genetics ; },
abstract = {Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutation efficiency in fibroblasts was as high as 87.5% in pig and 78.9% in buffalo. We then obtained single-cell clones with mutations at the specific sites of the GDF8 gene by screening with G418 in fibroblasts of pig and buffalo. In addition, the frequencies of Cas9/gRNA-mediated mutations were at 36 and 25% in the intracytoplasmic sperm injection embryos of pig and in vitro fertilization embryos of buffalo, respectively. Our work demonstrates that the Cas9/gRNA system is a highly efficient and fast tool for genome editing in cultured cells and embryos of Debao pig and swamp buffalo. These results can be helpful for the establishment of a new animal strain that can generate more meat.},
}

Animals
*Animals, Genetically Modified
Buffaloes/genetics
CRISPR-Cas Systems/*genetics
Cells, Cultured
Fertilization in Vitro
Fibroblasts
Gene Editing/*methods
Mutation
Myostatin/deficiency/*genetics
Phenotype
Swine/genetics

@article {pmid27916442,
year = {2016},
author = {Yasuda, H and Kim, E and Reza, AM and Kim, JH},
title = {A highly efficient method for enriching TALEN or CRISPR/Cas9-edited mutant cells.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {43},
number = {12},
pages = {705-708},
doi = {10.1016/j.jgg.2016.10.006},
pmid = {27916442},
issn = {1673-8527},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Separation/*methods ; *Gene Editing ; HCT116 Cells ; Humans ; Transcription Activator-Like Effector Nucleases/*metabolism ; },
}

CRISPR-Cas Systems/*genetics
Cell Separation/*methods
*Gene Editing
HCT116 Cells
Humans
Transcription Activator-Like Effector Nucleases/*metabolism

@article {pmid27543262,
year = {2016},
author = {Xu, R and Yang, Y and Qin, R and Li, H and Qiu, C and Li, L and Wei, P and Yang, J},
title = {Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {43},
number = {8},
pages = {529-532},
doi = {10.1016/j.jgg.2016.07.003},
pmid = {27543262},
issn = {1673-8527},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Oryza/*anatomy & histology/*genetics ; Whole Grains/*anatomy & histology/*genetics ; },
}

CRISPR-Cas Systems/*genetics
*Gene Editing
Oryza/*anatomy & histology/*genetics
Whole Grains/*anatomy & histology/*genetics

@article {pmid27222091,
year = {2016},
author = {Fu, Y and Rocha, PP and Luo, VM and Raviram, R and Deng, Y and Mazzoni, EO and Skok, JA},
title = {CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci.},
journal = {Nature communications},
volume = {7},
number = {},
pages = {11707},
doi = {10.1038/ncomms11707},
pmid = {27222091},
issn = {2041-1723},
support = {P30 CA016087/CA/NCI NIH HHS/United States ; R01 GM112192/GM/NIGMS NIH HHS/United States ; K99 GM117302/GM/NIGMS NIH HHS/United States ; R01 GM086852/GM/NIGMS NIH HHS/United States ; R01 HD079682/HD/NICHD NIH HHS/United States ; },
mesh = {3T3 Cells ; Animals ; *Bacterial Proteins ; *CRISPR-Cas Systems ; *Capsid Proteins ; DNA, Satellite/analysis ; *Endonucleases ; HEK293 Cells ; Humans ; Mice ; Optical Imaging/*methods ; Physical Chromosome Mapping ; *RNA, Guide ; },
abstract = {Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.},
}

3T3 Cells
Animals
*Bacterial Proteins
*CRISPR-Cas Systems
*Capsid Proteins
DNA, Satellite/analysis
*Endonucleases
HEK293 Cells
Humans
Mice
Optical Imaging/*methods
Physical Chromosome Mapping
*RNA, Guide

@article {pmid30228869,
year = {2018},
author = {Reimer, KA and Neugebauer, KM},
title = {Blood Relatives: Splicing Mechanisms underlying Erythropoiesis in Health and Disease.},
journal = {F1000Research},
volume = {7},
number = {},
pages = {},
doi = {10.12688/f1000research.15442.1},
pmid = {30228869},
issn = {2046-1402},
abstract = {During erythropoiesis, hematopoietic stem and progenitor cells transition to erythroblasts en route to terminal differentiation into enucleated red blood cells. Transcriptome-wide changes underlie distinct morphological and functional characteristics at each cell division during this process. Many studies of gene expression have historically been carried out in erythroblasts, and the biogenesis of β-globin mRNA-the most highly expressed transcript in erythroblasts-was the focus of many seminal studies on the mechanisms of pre-mRNA splicing. We now understand that pre-mRNA splicing plays an important role in shaping the transcriptome of developing erythroblasts. Recent advances have provided insight into the role of alternative splicing and intron retention as important regulatory mechanisms of erythropoiesis. However, dysregulation of splicing during erythropoiesis is also a cause of several hematological diseases, including β-thalassemia and myelodysplastic syndromes. With a growing understanding of the role that splicing plays in these diseases, we are well poised to develop gene-editing treatments. In this review, we focus on changes in the developing erythroblast transcriptome caused by alternative splicing, the molecular basis of splicing-related blood diseases, and therapeutic advances in disease treatment using CRISPR/Cas9 gene editing.},
}

@article {pmid30026227,
year = {2018},
author = {Grevet, JD and Lan, X and Hamagami, N and Edwards, CR and Sankaranarayanan, L and Ji, X and Bhardwaj, SK and Face, CJ and Posocco, DF and Abdulmalik, O and Keller, CA and Giardine, B and Sidoli, S and Garcia, BA and Chou, ST and Liebhaber, SA and Hardison, RC and Shi, J and Blobel, GA},
title = {Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells.},
journal = {Science (New York, N.Y.)},
volume = {361},
number = {6399},
pages = {285-290},
doi = {10.1126/science.aao0932},
pmid = {30026227},
issn = {1095-9203},
support = {R37 DK058044/DK/NIDDK NIH HHS/United States ; R56 DK065806/DK/NIDDK NIH HHS/United States ; R01 HL119479/HL/NHLBI NIH HHS/United States ; R01 GM110174/GM/NIGMS NIH HHS/United States ; U54 HG006998/HG/NHGRI NIH HHS/United States ; R01 AI118891/AI/NIAID NIH HHS/United States ; T32 GM008216/GM/NIGMS NIH HHS/United States ; R01 HL065449/HL/NHLBI NIH HHS/United States ; R01 MD009124/MD/NIMHD NIH HHS/United States ; F30 DK107055/DK/NIDDK NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/drug therapy/*genetics ; CRISPR-Cas Systems ; Carrier Proteins/*genetics/metabolism ; Cell Line ; Erythroid Cells/*metabolism ; Fetal Hemoglobin/*genetics ; *Gene Expression Regulation ; Genetic Testing ; Humans ; Molecular Targeted Therapy ; Nuclear Proteins/*genetics/metabolism ; RNA, Guide ; eIF-2 Kinase/*genetics/metabolism ; },
abstract = {Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of β-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain-focused CRISPR-Cas9-based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.},
}

Anemia, Sickle Cell/drug therapy/*genetics
CRISPR-Cas Systems
Carrier Proteins/*genetics/metabolism
Cell Line
Erythroid Cells/*metabolism
Fetal Hemoglobin/*genetics
*Gene Expression Regulation
Genetic Testing
Humans
Molecular Targeted Therapy
Nuclear Proteins/*genetics/metabolism
RNA, Guide
eIF-2 Kinase/*genetics/metabolism

@article {pmid29735716,
year = {2018},
author = {Champer, J and Liu, J and Oh, SY and Reeves, R and Luthra, A and Oakes, N and Clark, AG and Messer, PW},
title = {Reducing resistance allele formation in CRISPR gene drive.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {21},
pages = {5522-5527},
doi = {10.1073/pnas.1720354115},
pmid = {29735716},
issn = {1091-6490},
support = {R21 AI130635/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Disease Resistance/*genetics ; Drosophila Proteins/*genetics ; Drosophila melanogaster/*genetics ; *Gene Drive Technology ; Genetics, Population ; Germ Cells ; Mutation ; RNA, Guide/*genetics ; RNA-Binding Proteins ; },
abstract = {CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive in Drosophila melanogaster We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that the nanos promoter significantly lowers somatic Cas9 expression compared with the vasa promoter, suggesting that nanos provides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.},
}

Alleles
Animals
CRISPR-Cas Systems/*genetics
Disease Resistance/*genetics
Drosophila Proteins/*genetics
Drosophila melanogaster/*genetics
*Gene Drive Technology
Genetics, Population
Germ Cells
Mutation
RNA, Guide/*genetics
RNA-Binding Proteins

@article {pmid29626478,
year = {2018},
author = {He, J and Zhang, W and Li, A and Chen, F and Luo, R},
title = {Knockout of NCOA5 impairs proliferation and migration of hepatocellular carcinoma cells by suppressing epithelial-to-mesenchymal transition.},
journal = {Biochemical and biophysical research communications},
volume = {500},
number = {2},
pages = {177-183},
doi = {10.1016/j.bbrc.2018.04.017},
pmid = {29626478},
issn = {1090-2104},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Carcinoma, Hepatocellular/genetics/*pathology ; Cell Line, Tumor ; *Cell Movement/genetics ; Cell Proliferation/genetics ; *Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Neoplastic ; *Gene Knockout Techniques ; Humans ; Liver Neoplasms/genetics/*pathology ; Nuclear Receptor Coactivators/*metabolism ; Spheroids, Cellular/metabolism/pathology ; },
abstract = {Nuclear receptor coactivator 5 (NCOA5) plays important roles in the development of a variety of malignancies. However, the underlying mechanisms remain obscure. In this study, we successfully generated the NCOA5 knockout hepatocellular carcinoma (HCC) cells by CRISPR/Cas9 - mediated genome editing and found that knockout of NCOA5 inhibited the proliferation and tumor microsphere formation of HCC cells significantly. Moreover, the migration ability of NCOA5 knockout HCC cells declined. Mechanistic analyses indicated that knockout of NCOA5 can suppress the epithelial - mesenchymal transition (EMT) in HCC cells. In conclusion, our findings provide a mechanistic insight into the role of NCOA5 in HCC progression.},
}

Base Sequence
CRISPR-Cas Systems/genetics
Carcinoma, Hepatocellular/genetics/*pathology
Cell Line, Tumor
*Cell Movement/genetics
Cell Proliferation/genetics
*Epithelial-Mesenchymal Transition/genetics
Gene Expression Regulation, Neoplastic
*Gene Knockout Techniques
Humans
Liver Neoplasms/genetics/*pathology
Nuclear Receptor Coactivators/*metabolism
Spheroids, Cellular/metabolism/pathology

@article {pmid29180615,
year = {2017},
author = {Burguera, D and Marquez, Y and Racioppi, C and Permanyer, J and Torres-Méndez, A and Esposito, R and Albuixech-Crespo, B and Fanlo, L and D'Agostino, Y and Gohr, A and Navas-Perez, E and Riesgo, A and Cuomo, C and Benvenuto, G and Christiaen, LA and Martí, E and D'Aniello, S and Spagnuolo, A and Ristoratore, F and Arnone, MI and Garcia-Fernàndez, J and Irimia, M},
title = {Evolutionary recruitment of flexible Esrp-dependent splicing programs into diverse embryonic morphogenetic processes.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1799},
doi = {10.1038/s41467-017-01961-y},
pmid = {29180615},
issn = {2041-1723},
mesh = {Animals ; Biological Evolution ; CRISPR-Cas Systems ; Embryonic Development/*genetics ; Epithelial-Mesenchymal Transition/*physiology ; Exons/physiology ; Female ; Gene Expression Regulation, Developmental/physiology ; Gene Knockdown Techniques ; Lancelets ; Male ; Mutation ; RNA Splicing/*physiology ; RNA-Binding Proteins/genetics/*physiology ; Sequence Homology, Amino Acid ; Signal Transduction/genetics ; Strongylocentrotus purpuratus ; Urochordata ; Zebrafish ; },
abstract = {Epithelial-mesenchymal interactions are crucial for the development of numerous animal structures. Thus, unraveling how molecular tools are recruited in different lineages to control interplays between these tissues is key to understanding morphogenetic evolution. Here, we study Esrp genes, which regulate extensive splicing programs and are essential for mammalian organogenesis. We find that Esrp homologs have been independently recruited for the development of multiple structures across deuterostomes. Although Esrp is involved in a wide variety of ontogenetic processes, our results suggest ancient roles in non-neural ectoderm and regulating specific mesenchymal-to-epithelial transitions in deuterostome ancestors. However, consistent with the extensive rewiring of Esrp-dependent splicing programs between phyla, most developmental defects observed in vertebrate mutants are related to other types of morphogenetic processes. This is likely connected to the origin of an event in Fgfr, which was recruited as an Esrp target in stem chordates and subsequently co-opted into the development of many novel traits in vertebrates.},
}

Animals
Biological Evolution
CRISPR-Cas Systems
Embryonic Development/*genetics
Epithelial-Mesenchymal Transition/*physiology
Exons/physiology
Female
Gene Expression Regulation, Developmental/physiology
Gene Knockdown Techniques
Lancelets
Male
Mutation
RNA Splicing/*physiology
RNA-Binding Proteins/genetics/*physiology
Sequence Homology, Amino Acid
Signal Transduction/genetics
Strongylocentrotus purpuratus
Urochordata
Zebrafish

@article {pmid29123204,
year = {2017},
author = {Huai, C and Li, G and Yao, R and Zhang, Y and Cao, M and Kong, L and Jia, C and Yuan, H and Chen, H and Lu, D and Huang, Q},
title = {Structural insights into DNA cleavage activation of CRISPR-Cas9 system.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1375},
doi = {10.1038/s41467-017-01496-2},
pmid = {29123204},
issn = {2041-1723},
mesh = {Bacterial Proteins/*chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *DNA Cleavage ; Endonucleases/*chemistry/genetics/metabolism ; Molecular Dynamics Simulation ; Mutation ; Protein Domains ; RNA, Guide/chemistry/metabolism ; },
abstract = {CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease domains. However, structural information regarding the DNA cleavage-activating state of two nuclease domains remains sparse. Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA cleavage site. Compared with two known HNH states, our structure shows that the HNH active site moves toward the cleavage site by about 25 and 13 Å, respectively. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavage-activating architecture of Cas9.},
}

Bacterial Proteins/*chemistry/genetics/metabolism
*CRISPR-Cas Systems
Cryoelectron Microscopy
*DNA Cleavage
Endonucleases/*chemistry/genetics/metabolism
Molecular Dynamics Simulation
Mutation
Protein Domains
RNA, Guide/chemistry/metabolism

@article {pmid28532714,
year = {2017},
author = {},
title = {A race to bring CRISPR to the clinic.},
journal = {EBioMedicine},
volume = {19},
number = {},
pages = {1},
doi = {10.1016/j.ebiom.2017.05.014},
pmid = {28532714},
issn = {2352-3964},
mesh = {CRISPR-Cas Systems/*genetics ; Clinical Medicine/*trends ; HIV/genetics/pathogenicity ; Humans ; Receptors, Antigen, T-Cell/genetics/*therapeutic use ; },
}

CRISPR-Cas Systems/*genetics
Clinical Medicine/*trends
HIV/genetics/pathogenicity
Humans
Receptors, Antigen, T-Cell/genetics/*therapeutic use

@article {pmid30226584,
year = {2018},
author = {Liu, Q and Fan, D and Adah, D and Wu, Z and Liu, R and Yan, QT and Zhang, Y and Du, ZY and Wang, D and Li, Y and Bao, SY and Liu, LP},
title = {CRISPR/Cas9‑mediated hypoxia inducible factor‑1α knockout enhances the antitumor effect of transarterial embolization in hepatocellular carcinoma.},
journal = {Oncology reports},
volume = {},
number = {},
pages = {},
doi = {10.3892/or.2018.6667},
pmid = {30226584},
issn = {1791-2431},
abstract = {Transarterial embolization (TAE) is a palliative option commonly used for the treatment of advanced, unresectable hepatocellular carcinoma (HCC). However, patient prognosis in regards to overall survival has not improved with this method, mainly due to hypoxia‑inducible factor‑1α (HIF‑1α)‑induced angiogenesis and invasiveness. Thus, it is hypothesized that HIF‑1α may be an ideal knockout target for the treatment of HCC in combination with TAE. Thus, in the present study, HIF‑1α knockout was conducted in human liver cancer SMMC‑7721 cells and a xenograft HCC model was established using a lentivirus‑mediated CRISPR/Cas system (LV‑Cas) with small guide RNA‑721 (LV‑H721). Furthermore, hepatic artery ligation (HAL) was used to mimic human transarterial chemoembolization in mice. The results revealed that HIF‑1α was highly expressed in both HCC patient tissues and SMMC‑7721‑induced tumor tissues. The HIF‑1α knockout in SMMC‑7721 cells significantly suppressed cell invasiveness and migration, and induced cell apoptosis under CoCl2‑mimicking hypoxic conditions. Compared with the control groups, HAL + LV‑H721 inhibited SMMC‑7721 tumor growth in orthotopic HCC and markedly prolonged the survival of HCC‑bearing mice, which was accompanied by a lower CD31 expression (tumor angiogenesis) and increased apoptosis in the tumor cells. These findings demonstrated a valuable antitumor synergism in combining CRISPR/Cas9‑mediated HIF‑1α knockout with TAE in mice and highlighted the possibility that HIF‑1α may be an effective therapeutic knockout target in combination with TAE for HCC treatment.},
}

@article {pmid30224156,
year = {2018},
author = {Marzec, M and Hensel, G},
title = {Targeted Base Editing Systems Are Available for Plants.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2018.08.011},
pmid = {30224156},
issn = {1878-4372},
abstract = {Use of RNA-guided endonucleases for targeted genome editing is one of the most important breakthrough discoveries of the 21st century. Recent studies have described modifications of this precise base editing technique that open up a new dimension to plant genome editing.},
}

@article {pmid30045985,
year = {2018},
author = {Pautasso, S and Galitska, G and Dell'Oste, V and Biolatti, M and Cagliani, R and Forni, D and De Andrea, M and Gariglio, M and Sironi, M and Landolfo, S},
title = {Strategy of Human Cytomegalovirus To Escape Interferon Beta-Induced APOBEC3G Editing Activity.},
journal = {Journal of virology},
volume = {92},
number = {19},
pages = {},
doi = {10.1128/JVI.01224-18},
pmid = {30045985},
issn = {1098-5514},
mesh = {APOBEC-3G Deaminase/*genetics/immunology ; CRISPR-Cas Systems ; Cell Line ; Computational Biology ; Cytomegalovirus/*genetics/immunology ; Epithelial Cells/immunology/virology ; Fibroblasts/immunology/virology ; Foreskin/cytology ; Gene Expression Regulation ; Gene Knockout Techniques ; *Genome, Viral ; HEK293 Cells ; Human Umbilical Vein Endothelial Cells/immunology/virology ; Humans ; *Immune Evasion ; Immunity, Innate ; Interferon-beta/*genetics/immunology ; Male ; Mutagenesis ; Open Reading Frames ; Primary Cell Culture ; Signal Transduction ; THP-1 Cells ; Virus Replication ; },
abstract = {The apolipoprotein B editing enzyme catalytic subunit 3 (APOBEC3) is a family of DNA cytosine deaminases that mutate and inactivate viral genomes by single-strand DNA editing, thus providing an innate immune response against a wide range of DNA and RNA viruses. In particular, APOBEC3A (A3A), a member of the APOBEC3 family, is induced by human cytomegalovirus (HCMV) in decidual tissues where it efficiently restricts HCMV replication, thereby acting as an intrinsic innate immune effector at the maternal-fetal interface. However, the widespread incidence of congenital HCMV infection implies that HCMV has evolved to counteract APOBEC3-induced mutagenesis through mechanisms that still remain to be fully established. Here, we have assessed gene expression and deaminase activity of various APOBEC3 gene family members in HCMV-infected primary human foreskin fibroblasts (HFFs). Specifically, we show that APOBEC3G (A3G) gene products and, to a lesser degree, those of A3F but not of A3A, are upregulated in HCMV-infected HFFs. We also show that HCMV-mediated induction of A3G expression is mediated by interferon beta (IFN-β), which is produced early during HCMV infection. However, knockout or overexpression of A3G does not affect HCMV replication, indicating that A3G is not a restriction factor for HCMV. Finally, through a bioinformatics approach, we show that HCMV has evolved mutational robustness against IFN-β by limiting the presence of A3G hot spots in essential open reading frames (ORFs) of its genome. Overall, our findings uncover a novel immune evasion strategy by HCMV with profound implications for HCMV infections.IMPORTANCE APOBEC3 family of proteins plays a pivotal role in intrinsic immunity defense mechanisms against multiple viral infections, including retroviruses, through the deamination activity. However, the currently available data on APOBEC3 editing mechanisms upon HCMV infection remain unclear. In the present study, we show that particularly the APOBEC3G (A3G) member of the deaminase family is strongly induced upon infection with HCMV in fibroblasts and that its upregulation is mediated by IFN-β. Furthermore, we were able to demonstrate that neither A3G knockout nor A3G overexpression appears to modulate HCMV replication, indicating that A3G does not inhibit HCMV replication. This may be explained by HCMV escape strategy from A3G activity through depletion of the preferred nucleotide motifs (hot spots) from its genome. The results may shed light on antiviral potential of APOBEC3 activity during HCMV infection, as well as the viral counteracting mechanisms under A3G-mediated selective pressure.},
}

APOBEC-3G Deaminase/*genetics/immunology
CRISPR-Cas Systems
Cell Line
Computational Biology
Cytomegalovirus/*genetics/immunology
Epithelial Cells/immunology/virology
Fibroblasts/immunology/virology
Foreskin/cytology
Gene Expression Regulation
Gene Knockout Techniques
*Genome, Viral
HEK293 Cells
Human Umbilical Vein Endothelial Cells/immunology/virology
Humans
*Immune Evasion
Immunity, Innate
Interferon-beta/*genetics/immunology
Male
Mutagenesis
Open Reading Frames
Primary Cell Culture
Signal Transduction
THP-1 Cells
Virus Replication

@article {pmid29967137,
year = {2018},
author = {Shao, J and Wang, M and Yu, G and Zhu, S and Yu, Y and Heng, BC and Wu, J and Ye, H},
title = {Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {29},
pages = {E6722-E6730},
doi = {10.1073/pnas.1802448115},
pmid = {29967137},
issn = {1091-6490},
mesh = {Basic Helix-Loop-Helix Transcription Factors/*biosynthesis/genetics ; *CRISPR-Cas Systems ; *Cell Differentiation ; *Cellular Reprogramming ; *Epigenesis, Genetic ; HEK293 Cells ; HeLa Cells ; Humans ; *Light ; Nerve Tissue Proteins/*biosynthesis/genetics ; Neurons/cytology/*metabolism ; Optogenetics/methods ; Synthetic Biology ; },
abstract = {The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.},
}

Basic Helix-Loop-Helix Transcription Factors/*biosynthesis/genetics
*CRISPR-Cas Systems
*Cell Differentiation
*Cellular Reprogramming
*Epigenesis, Genetic
HEK293 Cells
HeLa Cells
Humans
*Light
Nerve Tissue Proteins/*biosynthesis/genetics
Neurons/cytology/*metabolism
Optogenetics/methods
Synthetic Biology

@article {pmid29614006,
year = {2018},
author = {Yajima, M and Ikuta, K and Kanda, T},
title = {Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells.},
journal = {Viruses},
volume = {10},
number = {4},
pages = {},
doi = {10.3390/v10040171},
pmid = {29614006},
issn = {1999-4915},
mesh = {*CRISPR-Cas Systems ; Chromosomes, Artificial, Bacterial ; *Cloning, Molecular ; DNA, Viral ; *Epstein-Barr Virus Infections ; *Genome, Viral ; Herpesvirus 4, Human/*genetics ; Homologous Recombination ; Humans ; Plasmids/genetics ; Sequence Analysis, DNA ; Transgenes ; Virus Integration ; *Virus Latency ; Whole Genome Sequencing ; },
abstract = {Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically.},
}

*CRISPR-Cas Systems
Chromosomes, Artificial, Bacterial
*Cloning, Molecular
DNA, Viral
*Epstein-Barr Virus Infections
*Genome, Viral
Herpesvirus 4, Human/*genetics
Homologous Recombination
Humans
Plasmids/genetics
Sequence Analysis, DNA
Transgenes
Virus Integration
*Virus Latency
Whole Genome Sequencing

@article {pmid29438322,
year = {2018},
author = {Chang, P and Yao, Y and Tang, N and Sadeyen, JR and Sealy, J and Clements, A and Bhat, S and Munir, M and Bryant, JE and Iqbal, M},
title = {The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus.},
journal = {Viruses},
volume = {10},
number = {2},
pages = {},
doi = {10.3390/v10020081},
pmid = {29438322},
issn = {1999-4915},
support = {BB/E01111X/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L018853/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007032//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P016472/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *DNA End-Joining Repair ; Gene Knock-In Techniques ; Genes, Reporter ; Genetic Vectors/genetics ; *Homologous Recombination ; INDEL Mutation ; Influenza A virus/genetics/*immunology ; Influenza Vaccines/immunology ; Integrases/*metabolism ; Mardivirus/genetics/*immunology ; RNA, Guide ; },
abstract = {Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.},
}

Animals
*CRISPR-Cas Systems
Cell Line
*DNA End-Joining Repair
Gene Knock-In Techniques
Genes, Reporter
Genetic Vectors/genetics
*Homologous Recombination
INDEL Mutation
Influenza A virus/genetics/*immunology
Influenza Vaccines/immunology
Integrases/*metabolism
Mardivirus/genetics/*immunology
RNA, Guide

@article {pmid29295784,
year = {2018},
author = {Kawabe, Y and Komatsu, S and Komatsu, S and Murakami, M and Ito, A and Sakuma, T and Nakamura, T and Yamamoto, T and Kamihira, M},
title = {Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems.},
journal = {Journal of bioscience and bioengineering},
volume = {125},
number = {5},
pages = {599-605},
doi = {10.1016/j.jbiosc.2017.12.003},
pmid = {29295784},
issn = {1347-4421},
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; Gene Knock-In Techniques/*methods ; Genes, Immunoglobulin ; Genetic Loci ; Hypoxanthine Phosphoribosyltransferase/*genetics ; Immunoglobulin Fc Fragments/*genetics ; Mutagenesis, Insertional/*methods ; Single-Chain Antibodies/*genetics ; Transgenes ; },
abstract = {Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.},
}

Animals
CHO Cells
*CRISPR-Cas Systems
Cricetinae
Cricetulus
Gene Knock-In Techniques/*methods
Genes, Immunoglobulin
Genetic Loci
Hypoxanthine Phosphoribosyltransferase/*genetics
Immunoglobulin Fc Fragments/*genetics
Mutagenesis, Insertional/*methods
Single-Chain Antibodies/*genetics
Transgenes

@article {pmid29269155,
year = {2018},
author = {Tang, N and Zhang, Y and Pedrera, M and Chang, P and Baigent, S and Moffat, K and Shen, Z and Nair, V and Yao, Y},
title = {A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system.},
journal = {Vaccine},
volume = {36},
number = {5},
pages = {716-722},
doi = {10.1016/j.vaccine.2017.12.025},
pmid = {29269155},
issn = {1873-2518},
support = {BB/P016472/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L014262/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Chick Embryo ; Chickens ; Fibroblasts ; Gene Editing ; Gene Expression ; Gene Knock-In Techniques ; Gene Order ; Gene Targeting ; Genes, Reporter ; Genetic Engineering ; Genetic Vectors/*genetics ; Herpesvirus 1, Meleagrid/*genetics ; Mice ; Vaccines, Synthetic/*genetics/*immunology ; Virus Replication ; },
abstract = {Herpesvirus of turkeys (HVT) has been successfully used as live vaccine against Marek's disease (MD) worldwide for more than 40 years either alone or in combination with other serotypes. HVT is also widely used as a vector platform for generation of recombinant vaccines against a number of avian diseases such as infectious bursal disease (IBD), Newcastle disease (ND) and avian influenza (AI) using conventional recombination methods or recombineering tools on cloned viral genomes. In the present study, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of generating HVT recombinants expressing VP2 protein of IBDV. This approach offers an efficient method to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines.},
}

Animals
*CRISPR-Cas Systems
Chick Embryo
Chickens
Fibroblasts
Gene Editing
Gene Expression
Gene Knock-In Techniques
Gene Order
Gene Targeting
Genes, Reporter
Genetic Engineering
Genetic Vectors/*genetics
Herpesvirus 1, Meleagrid/*genetics
Mice
Vaccines, Synthetic/*genetics/*immunology
Virus Replication

@article {pmid29187729,
year = {2017},
author = {Sun, N and Yu, X and Li, F and Liu, D and Suo, S and Chen, W and Chen, S and Song, L and Green, CD and McDermott, J and Shen, Q and Jing, N and Han, JJ},
title = {Inference of differentiation time for single cell transcriptomes using cell population reference data.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1856},
doi = {10.1038/s41467-017-01860-2},
pmid = {29187729},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle/genetics ; Cell Differentiation ; Cell Division/genetics ; Computational Biology/methods ; Gene Regulatory Networks ; High-Throughput Nucleotide Sequencing/methods ; Mice ; Mice, Knockout ; Mouse Embryonic Stem Cells/cytology/*physiology ; Proto-Oncogene Proteins c-fyn/genetics ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/*methods ; Smad1 Protein/genetics ; *Transcriptome ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Single-cell RNA sequencing (scRNA-seq) is a powerful method for dissecting intercellular heterogeneity during development. Conventional trajectory analysis provides only a pseudotime of development, and often discards cell-cycle events as confounding factors. Here using matched cell population RNA-seq (cpRNA-seq) as a reference, we developed an "iCpSc" package for integrative analysis of cpRNA-seq and scRNA-seq data. By generating a computational model for reference "biological differentiation time" using cell population data and applying it to single-cell data, we unbiasedly associated cell-cycle checkpoints to the internal molecular timer of single cells. Through inferring a network flow from cpRNA-seq to scRNA-seq data, we predicted a role of M phase in controlling the speed of neural differentiation of mouse embryonic stem cells, and validated it through gene knockout (KO) experiments. By linking temporally matched cpRNA-seq and scRNA-seq data, our approach provides an effective and unbiased approach for identifying developmental trajectory and timing-related regulatory events.},
}

Animals
CRISPR-Cas Systems
Cell Cycle/genetics
Cell Differentiation
Cell Division/genetics
Computational Biology/methods
Gene Regulatory Networks
High-Throughput Nucleotide Sequencing/methods
Mice
Mice, Knockout
Mouse Embryonic Stem Cells/cytology/*physiology
Proto-Oncogene Proteins c-fyn/genetics
Sequence Analysis, RNA/methods
Single-Cell Analysis/*methods
Smad1 Protein/genetics
*Transcriptome
Tumor Suppressor Protein p53/genetics

@article {pmid28432338,
year = {2017},
author = {Ludman, M and Burgyán, J and Fátyol, K},
title = {Crispr/Cas9 Mediated Inactivation of Argonaute 2 Reveals its Differential Involvement in Antiviral Responses.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {1010},
doi = {10.1038/s41598-017-01050-6},
pmid = {28432338},
issn = {2045-2322},
mesh = {Argonaute Proteins/*genetics ; CRISPR-Cas Systems ; Plant Diseases/genetics/*virology ; Plant Immunity ; Plant Proteins/genetics ; Plant Viruses/immunology/*pathogenicity ; Tobacco/genetics/immunology/*virology ; Tombusvirus/pathogenicity ; },
abstract = {RNA silencing constitutes an important antiviral mechanism in plants. Small RNA guided Argonaute proteins fulfill essential role in this process by acting as executors of viral restriction. Plants encode multiple Argonaute proteins of which several exhibit antiviral activities. A recent addition to this group is AGO2. Its involvement in antiviral responses is established predominantly by studies employing mutants of Arabidopsis thaliana. In the virological model plant, Nicotiana benthamiana, the contribution of AGO2 to antiviral immunity is much less certain due to the lack of appropriate genetic mutants. Previous studies employed various RNAi based tools to down-regulate AGO2 expression. However, these techniques have several disadvantages, especially in the context of antiviral RNA silencing. Here, we have utilized the CRISPR/Cas9 technology to inactivate the AGO2 gene of N. benthamiana. The ago2 plants exhibit differential sensitivities towards various viruses. AGO2 is a critical component of the plants' immune responses against PVX, TuMV and TCV. In contrast, AGO2 deficiency does not significantly influence the progression of tombusvirus and CMV infections. In summary, our work provides unequivocal proof for the virus-specific antiviral role of AGO2 in a plant species other than A. thaliana for the first time.},
}

Argonaute Proteins/*genetics
CRISPR-Cas Systems
Plant Diseases/genetics/*virology
Plant Immunity
Plant Proteins/genetics
Plant Viruses/immunology/*pathogenicity
Tobacco/genetics/immunology/*virology
Tombusvirus/pathogenicity

@article {pmid30222730,
year = {2018},
author = {Dorn, A and Röhrig, S and Papp, K and Schröpfer, S and Hartung, F and Knoll, A and Puchta, H},
title = {The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007674},
doi = {10.1371/journal.pgen.1007674},
pmid = {30222730},
issn = {1553-7404},
abstract = {Topoisomerase 3α, a class I topoisomerase, consists of a TOPRIM domain, an active centre and a variable number of zinc-finger domains (ZFDs) at the C-terminus, in multicellular organisms. Whereas the functions of the TOPRIM domain and the active centre are known, the specific role of the ZFDs is still obscure. In contrast to mammals where a knockout of TOP3α leads to lethality, we found that CRISPR/Cas induced mutants in Arabidopsis are viable but show growth retardation and meiotic defects, which can be reversed by the expression of the complete protein. However, complementation with AtTOP3α missing either the TOPRIM-domain or carrying a mutation of the catalytic tyrosine of the active centre leads to embryo lethality. Surprisingly, this phenotype can be overcome by the simultaneous removal of the ZFDs from the protein. In combination with a mutation of the nuclease AtMUS81, the TOP3α knockout proved to be also embryo lethal. Here, expression of TOP3α without ZFDs, and in particular without the conserved ZFD T1, leads to only a partly complementation in root growth-in contrast to the complete protein, that restores root length to mus81-1 mutant level. Expressing the E. coli resolvase RusA in this background, which is able to process Holliday junction (HJ)-like recombination intermediates, we could rescue this root growth defect. Considering all these results, we conclude that the ZFD T1 is specifically required for targeting the topoisomerase activity to HJ like recombination intermediates to enable their processing. In the case of an inactivated enzyme, this leads to cell death due to the masking of these intermediates, hindering their resolution by MUS81.},
}

@article {pmid29987047,
year = {2018},
author = {Verruto, J and Francis, K and Wang, Y and Low, MC and Greiner, J and Tacke, S and Kuzminov, F and Lambert, W and McCarren, J and Ajjawi, I and Bauman, N and Kalb, R and Hannum, G and Moellering, ER},
title = {Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {30},
pages = {E7015-E7022},
doi = {10.1073/pnas.1718193115},
pmid = {29987047},
issn = {1091-6490},
mesh = {*Acyl-CoA Oxidase/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; Light-Harvesting Protein Complexes/genetics/metabolism ; *Lipids/biosynthesis/genetics ; *Quantitative Trait, Heritable ; *Stramenopiles/genetics/metabolism ; },
abstract = {Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+ Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5'-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family.},
}

*Acyl-CoA Oxidase/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing
Light-Harvesting Protein Complexes/genetics/metabolism
*Lipids/biosynthesis/genetics
*Quantitative Trait, Heritable
*Stramenopiles/genetics/metabolism

@article {pmid29544035,
year = {2018},
author = {Borth, N and Hu, WS},
title = {Enhancing CHO by Systems Biotechnology.},
journal = {Biotechnology journal},
volume = {13},
number = {3},
pages = {e1800077},
doi = {10.1002/biot.201800077},
pmid = {29544035},
issn = {1860-7314},
mesh = {Animals ; *Biotechnology ; *CHO Cells ; CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; Gene Editing ; Genomic Instability ; *Systems Biology ; },
}

Animals
*Biotechnology
*CHO Cells
CRISPR-Cas Systems
Cricetinae
Cricetulus
Gene Editing
Genomic Instability
*Systems Biology

@article {pmid29374849,
year = {2018},
author = {Cai, M and Si, Y and Zhang, J and Tian, Z and Du, S},
title = {Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {20},
number = {2},
pages = {168-181},
doi = {10.1007/s10126-018-9794-8},
pmid = {29374849},
issn = {1436-2236},
support = {31230076//NSFC/ ; },
mesh = {Animals ; Argonaute Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing/methods ; HSP90 Heat-Shock Proteins/genetics/metabolism ; Luminescent Proteins/metabolism ; Muscles/*embryology ; Myosin Heavy Chains/genetics/metabolism ; Natronobacterium/genetics ; Sarcomeres/*genetics ; Zebrafish/embryology/*genetics ; },
abstract = {Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.},
}

Animals
Argonaute Proteins/genetics/metabolism
*CRISPR-Cas Systems
Gene Editing/methods
HSP90 Heat-Shock Proteins/genetics/metabolism
Luminescent Proteins/metabolism
Muscles/*embryology
Myosin Heavy Chains/genetics/metabolism
Natronobacterium/genetics
Sarcomeres/*genetics
Zebrafish/embryology/*genetics

@article {pmid29298051,
year = {2018},
author = {Ray, M and Lee, YW and Hardie, J and Mout, R and Yeşilbag Tonga, G and Farkas, ME and Rotello, VM},
title = {CRISPRed Macrophages for Cell-Based Cancer Immunotherapy.},
journal = {Bioconjugate chemistry},
volume = {29},
number = {2},
pages = {445-450},
doi = {10.1021/acs.bioconjchem.7b00768},
pmid = {29298051},
issn = {1520-4812},
support = {R01 EB014277/EB/NIBIB NIH HHS/United States ; R01 EB022641/EB/NIBIB NIH HHS/United States ; R01 GM077173/GM/NIGMS NIH HHS/United States ; T32 GM008515/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Humans ; Immunotherapy/methods ; Macrophages/immunology/*metabolism ; Mice ; Nanomedicine/methods ; Neoplasms/immunology/therapy ; Phagocytosis ; RAW 264.7 Cells ; Receptors, Immunologic/*genetics/immunology ; },
abstract = {We present here an integrated nanotechnology/biology strategy for cancer immunotherapy that uses arginine nanoparticles (ArgNPs) to deliver CRISPR-Cas9 gene editing machinery into cells to generate SIRP-α knockout macrophages. The NP system efficiently codelivers single guide RNA (sgRNA) and Cas9 protein required for editing to knock out the "don't eat me signal" in macrophages that prevents phagocytosis of cancer cells. Turning off this signal increased the innate phagocytic capabilities of the macrophages by 4-fold. This improved attack and elimination of cancer cells makes this strategy promising for the creation of "weaponized" macrophages for cancer immunotherapy.},
}

Animals
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing/*methods
Gene Knockout Techniques/methods
Humans
Immunotherapy/methods
Macrophages/immunology/*metabolism
Mice
Nanomedicine/methods
Neoplasms/immunology/therapy
Phagocytosis
RAW 264.7 Cells
Receptors, Immunologic/*genetics/immunology

@article {pmid29127285,
year = {2017},
author = {Shibata, M and Nishimasu, H and Kodera, N and Hirano, S and Ando, T and Uchihashi, T and Nureki, O},
title = {Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1430},
doi = {10.1038/s41467-017-01466-8},
pmid = {29127285},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*physiology ; Catalytic Domain ; Computer Systems ; DNA/chemistry/metabolism ; Enzyme Stability ; Microscopy, Atomic Force/*methods ; Microscopy, Video/*methods ; Models, Biological ; Models, Molecular ; RNA/chemistry/metabolism ; Thermodynamics ; },
abstract = {The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.},
}

CRISPR-Cas Systems/*physiology
Catalytic Domain
Computer Systems
DNA/chemistry/metabolism
Enzyme Stability
Microscopy, Atomic Force/*methods
Microscopy, Video/*methods
Models, Biological
Models, Molecular
RNA/chemistry/metabolism
Thermodynamics

@article {pmid29127284,
year = {2017},
author = {Harrington, LB and Paez-Espino, D and Staahl, BT and Chen, JS and Ma, E and Kyrpides, NC and Doudna, JA},
title = {A thermostable Cas9 with increased lifetime in human plasma.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1424},
doi = {10.1038/s41467-017-01408-4},
pmid = {29127284},
issn = {2041-1723},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacterial Proteins/administration & dosage/blood/*metabolism ; CRISPR-Cas Systems ; Endonucleases/administration & dosage/blood/*metabolism ; Enzyme Stability ; Gene Editing ; Geobacillus stearothermophilus/*enzymology ; Hot Temperature ; Humans ; Models, Molecular ; Protein Engineering ; Ribonucleoproteins/administration & dosage ; },
abstract = {CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas9 is active at temperatures up to 70 °C, compared to 45 °C for Streptococcus pyogenes Cas9 (SpyCas9), which expands the temperature range for CRISPR-Cas9 applications. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.},
}

Bacterial Proteins/administration & dosage/blood/*metabolism
CRISPR-Cas Systems
Endonucleases/administration & dosage/blood/*metabolism
Enzyme Stability
Gene Editing
Geobacillus stearothermophilus/*enzymology
Hot Temperature
Humans
Models, Molecular
Protein Engineering
Ribonucleoproteins/administration & dosage

@article {pmid29058367,
year = {2018},
author = {Aparicio, T and de Lorenzo, V and Martínez-García, E},
title = {CRISPR/Cas9-Based Counterselection Boosts Recombineering Efficiency in Pseudomonas putida.},
journal = {Biotechnology journal},
volume = {13},
number = {5},
pages = {e1700161},
doi = {10.1002/biot.201700161},
pmid = {29058367},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Mutation/genetics ; Plasmids/genetics ; *Pseudomonas putida/genetics/metabolism ; },
abstract = {While adoption of single-stranded DNA recombineering techniques has greatly eased genetic design of the platform strain Pseudomonas putida KT2440, available methods still produce the desired modifications/deletions at low frequencies. This makes isolation of mutants that do not display selectable or conspicuous phenotypes considerably difficult. To overcome this limitation, the authors have merged ssDNA recombineering with CRISPR/Cas9 technology in this bacterium for efficient killing of unmodified cells and thus non-phenotypic selection of bacteria bearing the mutations of interest. After incorporating the system into standardized pSEVA plasmids the authors tested its functional efficiency by targeting different types of changes that ranged from single nucleotide substitutions to one-gene deletions-to even the removal of the large flagellar cluster (≈69 kb). Simultaneous introduction of two independent gene deletions was tested as well. In all cases, directing the crRNA/Cas9 complexes toward non-modified, wild-type genomic sequences boosted dramatically the appearance of the mutants at stake in the absence of any phenotypic selection. The results presented here upgrade the engineering possibilities of the genome of this environmental bacterium (and possibly other Gram-negatives) to obtain modifications that are otherwise cumbersome to generate.},
}

*CRISPR-Cas Systems
*Gene Editing
Mutation/genetics
Plasmids/genetics
*Pseudomonas putida/genetics/metabolism

@article {pmid28826592,
year = {2017},
author = {Gruber, K},
title = {Genetic basis for response to cancer immunotherapy.},
journal = {The Lancet. Oncology},
volume = {18},
number = {9},
pages = {e521},
doi = {10.1016/S1470-2045(17)30627-7},
pmid = {28826592},
issn = {1474-5488},
mesh = {Biomarkers, Tumor/*genetics/immunology ; CRISPR-Cas Systems ; Databases, Genetic ; Drug Resistance, Neoplasm/genetics ; Gene Editing/methods ; Humans ; Immunotherapy/adverse effects/*methods ; Neoplasms/genetics/immunology/pathology/*therapy ; Treatment Outcome ; Tumor Escape/genetics ; Whole Exome Sequencing ; },
}

Biomarkers, Tumor/*genetics/immunology
CRISPR-Cas Systems
Databases, Genetic
Drug Resistance, Neoplasm/genetics
Gene Editing/methods
Humans
Immunotherapy/adverse effects/*methods
Neoplasms/genetics/immunology/pathology/*therapy
Treatment Outcome
Tumor Escape/genetics
Whole Exome Sequencing

@article {pmid28512121,
year = {2017},
author = {Courtier-Orgogozo, V and Morizot, B and Boëte, C},
title = {Agricultural pest control with CRISPR-based gene drive: time for public debate: Should we use gene drive for pest control?.},
journal = {EMBO reports},
volume = {18},
number = {6},
pages = {878-880},
doi = {10.15252/embr.201744205},
pmid = {28512121},
issn = {1469-3178},
mesh = {Agriculture/*methods ; CRISPR-Cas Systems/*genetics ; Ecosystem ; Pest Control/*methods ; },
}

Agriculture/*methods
CRISPR-Cas Systems/*genetics
Ecosystem
Pest Control/*methods

@article {pmid27256542,
year = {2016},
author = {Jiao, R and Gao, C},
title = {The CRISPR/Cas9 Genome Editing Revolution.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {43},
number = {5},
pages = {227-228},
doi = {10.1016/j.jgg.2016.05.004},
pmid = {27256542},
issn = {1673-8527},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods/*trends ; Genome/*genetics ; Genome, Plant/genetics ; Humans ; Mice ; },
}

Animals
CRISPR-Cas Systems/*genetics
Gene Editing/*methods/*trends
Genome/*genetics
Genome, Plant/genetics
Humans
Mice

@article {pmid30195321,
year = {2018},
author = {Karagyaur, MN and Rubtsov, YP and Vasiliev, PA and Tkachuk, VA},
title = {Practical Recommendations for Improving Efficiency and Accuracy of the CRISPR/Cas9 Genome Editing System.},
journal = {Biochemistry. Biokhimiia},
volume = {83},
number = {6},
pages = {629-642},
doi = {10.1134/S0006297918060020},
pmid = {30195321},
issn = {1608-3040},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Breaks ; DNA Repair ; Gene Editing/*methods ; Genetic Vectors/genetics/metabolism ; Humans ; RNA, Guide/chemistry/metabolism ; },
abstract = {CRISPR/Cas9 genome-editing system is a powerful, fairly accurate, and efficient tool for modifying genomic DNA. Despite obvious advantages, it is not devoid of certain drawbacks, such as propensity for introduction of additional nonspecific DNA breaks, insufficient activity against aneuploid genomes, and relative difficulty in delivering its components to cells. In this review, we focus on the difficulties that can limit the use of CRISPR/Cas9 and suggest a number of practical recommendations and information sources that will make it easier for the beginners to work with this outstanding technological achievement of the XXI century.},
}

CRISPR-Cas Systems/*genetics
DNA Breaks
DNA Repair
Gene Editing/*methods
Genetic Vectors/genetics/metabolism
Humans
RNA, Guide/chemistry/metabolism

@article {pmid29665362,
year = {2018},
author = {Ooga, M and Funaya, S and Hashioka, Y and Fujii, W and Naito, K and Suzuki, MG and Aoki, F},
title = {Chd9 mediates highly loosened chromatin structure in growing mouse oocytes.},
journal = {Biochemical and biophysical research communications},
volume = {500},
number = {3},
pages = {583-588},
doi = {10.1016/j.bbrc.2018.04.105},
pmid = {29665362},
issn = {1090-2104},
mesh = {Animals ; Base Sequence ; Blastocyst/metabolism ; CRISPR-Cas Systems/genetics ; Cell Proliferation ; Chromatin/*metabolism ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Green Fluorescent Proteins/metabolism ; Histones/metabolism ; Mice, Inbred C57BL ; Mice, Knockout ; Oocytes/*cytology/*metabolism ; Trans-Activators/deficiency/*metabolism ; },
abstract = {During oogenesis, oocytes prepare for embryonic development following fertilization. The mechanisms underlying this process are still unknown. Recently, it has been suggested that a loosened chromatin structure is involved in pluripotency and totipotency in embryonic stem (ES) cells and early preimplantation embryos, respectively. Here, we explored chromatin looseness in oocytes by fluorescence recovery after photobleaching (FRAP) using enhanced green fluorescent protein-tagged histone H2B. The results indicated that the chromatin in growing oocytes was already highly loosened to a level comparable to that in early preimplantation embryos. To elucidate the mechanism underlying the loosened chromatin structure in oocytes, we focused on chromodomain helicase DNA binding protein 9 (Chd9), which is highly expressed in growing oocytes. The oocytes from Chd9 knockout mice (Chd9-/-) generated using the CRISPR/Cas9 system exhibited a less loosened chromatin structure than that of wild-type mice, suggesting that Chd9 is involved in the loosened chromatin structure in growing oocytes. These results suggest that a loosened chromatin structure, which is mediated by Chd9, is a prerequisite for the acquisition of totipotency after fertilization.},
}

Animals
Base Sequence
Blastocyst/metabolism
CRISPR-Cas Systems/genetics
Cell Proliferation
Chromatin/*metabolism
Female
Gene Expression Profiling
Gene Expression Regulation, Developmental
Green Fluorescent Proteins/metabolism
Histones/metabolism
Mice, Inbred C57BL
Mice, Knockout
Oocytes/*cytology/*metabolism
Trans-Activators/deficiency/*metabolism

@article {pmid29405266,
year = {2018},
author = {Kodama, M and Yoshida, M and Endo, M and Kobayashi, T and Oike, A and Yasumasu, S and Nakamura, M},
title = {Nanos3 of the frog Rana rugosa: Molecular cloning and characterization.},
journal = {Development, growth & differentiation},
volume = {60},
number = {2},
pages = {112-120},
doi = {10.1111/dgd.12421},
pmid = {29405266},
issn = {1440-169X},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Cloning, Molecular ; DEAD-box RNA Helicases/analysis ; Female ; Germ Cells/*metabolism ; Imaging, Three-Dimensional ; Male ; Mice ; Ovary/cytology/*metabolism ; RNA, Messenger/genetics ; RNA-Binding Proteins/*genetics ; Ranidae/*embryology ; Testis/cytology/*metabolism ; },
abstract = {Nanos is expressed in the primordial germ cells (PGCs) and also the germ cells of a variety of organisms as diverse as Drosophila, medaka fish, Xenopus and mouse. In Nanos3-deficient mice, PGCs fail to incorporate into the gonad and the size of the testis and ovary is thereby dramatically reduced. To elucidate the role of Nanos in an amphibian species, we cloned Nanos3 cDNA from the testis of the R. rugosa frog. RT-PCR analysis showed strong expression of Nanos3 mRNA in the testis of adult R. rugosa frogs, but expression was not sexually dimorphic during gonadal differentiation. In Nanos3-knockdown tadpoles produced by the CRISPR/Cas9 system, the number of germ cells decreased dramatically in the gonads of both male and female tadpoles before sex determination and thereafter. This was confirmed by three dimensional imaging of wild-type and Nanos3 knockdown gonads using serial sections immunostained for Vasa, a marker specific to germ cells. Taken together, these results suggest that Nanos3 protein function is conserved between R. rugosa and mouse.},
}

Amino Acid Sequence
Animals
CRISPR-Cas Systems
Cloning, Molecular
DEAD-box RNA Helicases/analysis
Female
Germ Cells/*metabolism
Imaging, Three-Dimensional
Male
Mice
Ovary/cytology/*metabolism
RNA, Messenger/genetics
RNA-Binding Proteins/*genetics
Ranidae/*embryology
Testis/cytology/*metabolism

@article {pmid29274230,
year = {2017},
author = {Xu, XS and Gantz, VM and Siomava, N and Bier, E},
title = {CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity.},
journal = {eLife},
volume = {6},
number = {},
pages = {},
doi = {10.7554/eLife.30281},
pmid = {29274230},
issn = {2050-084X},
support = {Distinguished Investigator Award//Paul G. Allen Frontiers Group/International ; 1DP5OD023098/NH/NIH HHS/United States ; 1R01GM117321/NH/NIH HHS/United States ; P30 NS047101/NS/NINDS NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; DP5 OD023098/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila Proteins/*genetics ; Drosophila melanogaster/classification/*genetics/growth & development ; *Gene Expression Regulation, Developmental ; *Regulatory Sequences, Nucleic Acid ; Repressor Proteins/*genetics ; Species Specificity ; Wings, Animal/*growth & development/physiology ; },
abstract = {The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these 'in locus' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.},
}

Animals
*CRISPR-Cas Systems
Drosophila Proteins/*genetics
Drosophila melanogaster/classification/*genetics/growth & development
*Gene Expression Regulation, Developmental
*Regulatory Sequences, Nucleic Acid
Repressor Proteins/*genetics
Species Specificity
Wings, Animal/*growth & development/physiology

@article {pmid27795546,
year = {2016},
author = {Carrasquilla, M and Owusu, CK},
title = {A CRISPR outlook for apicomplexans.},
journal = {Nature reviews. Microbiology},
volume = {14},
number = {11},
pages = {668},
doi = {10.1038/nrmicro.2016.153},
pmid = {27795546},
issn = {1740-1534},
mesh = {*CRISPR-Cas Systems ; Gene Knockout Techniques/*methods ; Plasmodium berghei/*genetics ; Plasmodium falciparum/*genetics ; Toxoplasma/*genetics ; },
}

*CRISPR-Cas Systems
Gene Knockout Techniques/*methods
Plasmodium berghei/*genetics
Plasmodium falciparum/*genetics
Toxoplasma/*genetics

@article {pmid27694808,
year = {2016},
author = {Waldron, D},
title = {Genetic engineering: Allele-specific genome editing of disease loci.},
journal = {Nature reviews. Genetics},
volume = {17},
number = {11},
pages = {660},
doi = {10.1038/nrg.2016.131},
pmid = {27694808},
issn = {1471-0064},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Editing ; *Genetic Engineering ; Genetic Loci/*genetics ; Genome, Human/*genetics ; Humans ; Huntington Disease/*genetics ; Mutation/genetics ; },
}

Alleles
Animals
CRISPR-Cas Systems/genetics
*Gene Editing
*Genetic Engineering
Genetic Loci/*genetics
Genome, Human/*genetics
Humans
Huntington Disease/*genetics
Mutation/genetics

@article {pmid27291817,
year = {2016},
author = {Burgess, DJ},
title = {Technique: Genome editing for cell lineage tracing.},
journal = {Nature reviews. Genetics},
volume = {17},
number = {8},
pages = {435},
doi = {10.1038/nrg.2016.80},
pmid = {27291817},
issn = {1471-0064},
mesh = {CRISPR-Cas Systems/genetics ; Cell Lineage/*genetics ; DNA/genetics ; Gene Editing/*methods ; Humans ; Mutation/genetics ; RNA, Guide/genetics ; },
}

CRISPR-Cas Systems/genetics
Cell Lineage/*genetics
DNA/genetics
Gene Editing/*methods
Humans
Mutation/genetics
RNA, Guide/genetics

@article {pmid30209390,
year = {2018},
author = {Akcakaya, P and Bobbin, ML and Guo, JA and Malagon-Lopez, J and Clement, K and Garcia, SP and Fellows, MD and Porritt, MJ and Firth, MA and Carreras, A and Baccega, T and Seeliger, F and Bjursell, M and Tsai, SQ and Nguyen, NT and Nitsch, R and Mayr, LM and Pinello, L and Bohlooly-Y, M and Aryee, MJ and Maresca, M and Joung, JK},
title = {In vivo CRISPR editing with no detectable genome-wide off-target mutations.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-018-0500-9},
pmid = {30209390},
issn = {1476-4687},
abstract = {CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.},
}

@article {pmid30209206,
year = {2018},
author = {Cubbon, A and Ivancic-Bace, I and Bolt, EL},
title = {CRISPR-Cas Immunity, DNA repair and Genome Stability.},
journal = {Bioscience reports},
volume = {},
number = {},
pages = {},
doi = {10.1042/BSR20180457},
pmid = {30209206},
issn = {1573-4935},
abstract = {Co-opting of CRISPR-Cas "Interference" reactions for editing the genomes of eukaryotic and prokaryotic cells has highlighted crucial support roles for DNA repair systems that strive to maintain genome stability. As front-runners in genome editing that targets DNA, the class 2 CRISPR-Cas enzymes Cas9 and Cas12a rely on repair of DNA double-strand breaks (DDSBs) by host DNA repair enzymes, using mechanisms that vary in how well they are understood. Data is emerging about the identities of DNA repair enzymes that support genome editing in human cells. At the same time, it is becoming apparent that CRISPR-Cas systems functioning in their native environment, bacteria or archaea, also need DNA repair enzymes. In this short review we survey how DNA repair and CRISPR-Cas systems are inter-twined. We consider how understanding DNA repair and CRISPR-Cas interference reactions in nature might help improve the efficacy of genome editing procedures that utilize homologous or analogous systems in human and other cells.},
}

@article {pmid30072518,
year = {2018},
author = {Kupferschmidt, K},
title = {EU verdict on CRISPR crops dismays scientists.},
journal = {Science (New York, N.Y.)},
volume = {361},
number = {6401},
pages = {435-436},
doi = {10.1126/science.361.6401.435},
pmid = {30072518},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Crops, Agricultural/*genetics ; European Union ; Gene Editing/*legislation & jurisprudence ; Plants, Genetically Modified/*genetics ; },
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Crops, Agricultural/*genetics
European Union
Gene Editing/*legislation & jurisprudence
Plants, Genetically Modified/*genetics

@article {pmid27916995,
year = {2016},
author = {Wei, Y and Chen, Y and Qiu, Y and Zhao, H and Liu, G and Zhang, Y and Meng, Q and Wu, G and Chen, Y and Cai, X and Wang, H and Ying, H and Zhou, B and Liu, M and Li, D and Ding, Q},
title = {Prevention of Muscle Wasting by CRISPR/Cas9-mediated Disruption of Myostatin In Vivo.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {24},
number = {11},
pages = {1889-1891},
doi = {10.1038/mt.2016.192},
pmid = {27916995},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Muscle, Skeletal/physiopathology ; Myostatin/*metabolism ; Wasting Syndrome/genetics/therapy ; },
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Muscle, Skeletal/physiopathology
Myostatin/*metabolism
Wasting Syndrome/genetics/therapy

@article {pmid27916992,
year = {2016},
author = {Tremblay, JP and Iyombe-Engembe, JP and Duchêne, B and Ouellet, DL},
title = {Gene Editing for Duchenne Muscular Dystrophy Using the CRISPR/Cas9 Technology: The Importance of Fine-tuning the Approach.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {24},
number = {11},
pages = {1888-1889},
doi = {10.1038/mt.2016.191},
pmid = {27916992},
issn = {1525-0024},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dystrophin ; *Gene Editing ; Humans ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne/*genetics/therapy ; },
}

Animals
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Dystrophin
*Gene Editing
Humans
Mice
Mice, Inbred mdx
Muscular Dystrophy, Duchenne/*genetics/therapy

@article {pmid30208287,
year = {2018},
author = {Stanley, SY and Maxwell, KL},
title = {Phage-Encoded Anti-CRISPR Defenses.},
journal = {Annual review of genetics},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-genet-120417-031321},
pmid = {30208287},
issn = {1545-2948},
abstract = {The battle for survival between bacteria and bacteriophages (phages) is an arms race where bacteria develop defenses to protect themselves from phages and phages evolve counterstrategies to bypass these defenses. CRISPR-Cas adaptive immune systems represent a widespread mechanism by which bacteria protect themselves from phage infection. In response to CRISPR-Cas, phages have evolved protein inhibitors known as anti-CRISPRs. Here, we describe the discovery and mechanisms of action of anti-CRISPR proteins. We discuss the potential impact of anti-CRISPRs on bacterial evolution, speculate on their evolutionary origins, and contemplate the possible next steps in the CRISPR-Cas evolutionary arms race. We also touch on the impact of anti-CRISPRs on the development of CRISPR-Cas-based biotechnological tools. Expected final online publication date for the Annual Review of Genetics Volume 52 is November 23, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},
}

@article {pmid30205462,
year = {2018},
author = {Hidalgo-Cantabrana, C and Sanozky-Dawes, R and Barrangou, R},
title = {Insights into the Human Virome Using CRISPR Spacers from Microbiomes.},
journal = {Viruses},
volume = {10},
number = {9},
pages = {},
doi = {10.3390/v10090479},
pmid = {30205462},
issn = {1999-4915},
support = {support//North Carolina Agricultural Foundation/ ; internal support//North Carolina State University/ ; },
abstract = {Due to recent advances in next-generation sequencing over the past decade, our understanding of the human microbiome and its relationship to health and disease has increased dramatically. Yet, our insights into the human virome, and its interplay with important microbes that impact human health, is relatively limited. Prokaryotic and eukaryotic viruses are present throughout the human body, comprising a large and diverse population which influences several niches and impacts our health at various body sites. The presence of prokaryotic viruses like phages, has been documented at many different body sites, with the human gut being the richest ecological niche. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins constitute the adaptive immune system of bacteria, which prevents attack by invasive nucleic acid. CRISPR-Cas systems function by uptake and integration of foreign genetic element sequences into the CRISPR array, which constitutes a genomic archive of iterative vaccination events. Consequently, CRISPR spacers can be investigated to reconstruct interplay between viruses and bacteria, and metagenomic sequencing data can be exploited to provide insights into host-phage interactions within a niche. Here, we show how the CRISPR spacer content of commensal and pathogenic bacteria can be used to determine the evidence of their phage exposure. This framework opens new opportunities for investigating host-virus dynamics in metagenomic data, and highlights the need to dedicate more efforts for virome sampling and sequencing.},
}

@article {pmid29446747,
year = {2018},
author = {Zhang, Y and Sastre, D and Wang, F},
title = {CRISPR/Cas9 Genome Editing: A Promising Tool for Therapeutic Applications of Induced Pluripotent Stem Cells.},
journal = {Current stem cell research & therapy},
volume = {13},
number = {4},
pages = {243-251},
doi = {10.2174/1574888X13666180214124800},
pmid = {29446747},
issn = {2212-3946},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Humans ; Induced Pluripotent Stem Cells/*cytology ; },
abstract = {BACKGROUND: Induced pluripotent stem cells hold tremendous potential for biological and therapeutic applications. The development of efficient technologies for targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system is recently recognized as a powerful tool for editing DNA at specific loci.

OBJECTIVE: The ease of use of the CRISPR-Cas9 technology will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. More recently, this system was modified to repress (CRISPR interference, CRISPRi) or activate (CRISPR activation, CRISPRa) gene expression without alterations in the DNA, which amplified the scope of applications of CRISPR systems for stem cell biology.

RESULTS AND CONCLUSION: Here, we highlight latest advances of CRISPR-associated applications in human pluripotent stem cells. The challenges and future prospects of CRISPR-based systems for human research are also discussed.},
}

Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing/methods
Humans
Induced Pluripotent Stem Cells/*cytology

@article {pmid29051052,
year = {2018},
author = {Ali, Z and Eid, A and Ali, S and Mahfouz, MM},
title = {Pea early-browning virus-mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis.},
journal = {Virus research},
volume = {244},
number = {},
pages = {333-337},
doi = {10.1016/j.virusres.2017.10.009},
pmid = {29051052},
issn = {1872-7492},
mesh = {Arabidopsis/*genetics/metabolism ; Bacterial Proteins/administration & dosage/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/administration & dosage/*genetics/metabolism ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Loci ; *Genome, Plant ; Nuclear Localization Signals ; Plant Leaves/genetics/metabolism ; Plant Viruses/*genetics/metabolism ; Plants, Genetically Modified ; RNA, Guide/administration & dosage/*genetics/metabolism ; Tobacco/*genetics/metabolism ; },
abstract = {The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species. Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.},
}

Arabidopsis/*genetics/metabolism
Bacterial Proteins/administration & dosage/*genetics/metabolism
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Endonucleases/administration & dosage/*genetics/metabolism
Gene Editing/methods
Gene Transfer Techniques
Genetic Loci
*Genome, Plant
Nuclear Localization Signals
Plant Leaves/genetics/metabolism
Plant Viruses/*genetics/metabolism
Plants, Genetically Modified
RNA, Guide/administration & dosage/*genetics/metabolism
Tobacco/*genetics/metabolism

@article {pmid28456574,
year = {2018},
author = {Yuen, KS and Wang, ZM and Wong, NM and Zhang, ZQ and Cheng, TF and Lui, WY and Chan, CP and Jin, DY},
title = {Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9.},
journal = {Virus research},
volume = {244},
number = {},
pages = {296-303},
doi = {10.1016/j.virusres.2017.04.019},
pmid = {28456574},
issn = {1872-7492},
mesh = {Antineoplastic Agents/pharmacology ; Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/drug effects ; Cisplatin/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Viral/*genetics/metabolism ; Endonucleases/*genetics/metabolism ; Epithelial Cells/drug effects/pathology/virology ; Fluorouracil/pharmacology ; Gene Editing/*methods ; *Genome, Viral ; Herpesvirus 4, Human/drug effects/*genetics/growth & development/metabolism ; Humans ; Nasopharynx/drug effects/pathology/virology ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; Viral Load/drug effects ; Virus Latency/genetics ; Virus Replication ; },
abstract = {Epstein-Barr virus (EBV) infects more than 90% of the world's adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.},
}

Antineoplastic Agents/pharmacology
Bacterial Proteins/*genetics/metabolism
*CRISPR-Cas Systems
Cell Line, Tumor
Cell Survival/drug effects
Cisplatin/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats
DNA, Viral/*genetics/metabolism
Endonucleases/*genetics/metabolism
Epithelial Cells/drug effects/pathology/virology
Fluorouracil/pharmacology
Gene Editing/*methods
*Genome, Viral
Herpesvirus 4, Human/drug effects/*genetics/growth & development/metabolism
Humans
Nasopharynx/drug effects/pathology/virology
Plasmids/chemistry/metabolism
RNA, Guide/*genetics/metabolism
Viral Load/drug effects
Virus Latency/genetics
Virus Replication

@article {pmid28279800,
year = {2018},
author = {Li, Z and Bi, Y and Xiao, H and Sun, L and Ren, Y and Li, Y and Chen, C and Cun, W},
title = {CRISPR-Cas9 system-driven site-specific selection pressure on Herpes simplex virus genomes.},
journal = {Virus research},
volume = {244},
number = {},
pages = {286-295},
doi = {10.1016/j.virusres.2017.03.010},
pmid = {28279800},
issn = {1872-7492},
mesh = {Animals ; Bacterial Proteins/*genetics/metabolism ; Base Sequence ; *CRISPR-Cas Systems ; Cercopithecus aethiops ; Clustered Regularly Interspaced Short Palindromic Repeats ; Deoxyribonuclease I/genetics/metabolism ; Endonucleases/*genetics/metabolism ; Gene Editing/*methods ; Genes, Reporter ; *Genome, Viral ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Herpesvirus 1, Human/*genetics/metabolism ; Homologous Recombination ; Humans ; Lentivirus/genetics/metabolism ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; Selection, Genetic ; Vero Cells ; Virus Replication ; },
abstract = {The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system has been widely used for viral genome editing, transcription regulation and chromosomal localization in eukaryotic cells. In this study, a guide RNA (gRNA) that specifically recognizes HSV-1 viral genomes was used in the CRISPR-Cas9 system to inhibit viral replication. This inhibition could be achieved with both wild type Cas9 protein and Cas9 nickase (D10A). By targeting viral genomes containing sequences recognized by the gRNA, the CRISPR-Cas9 system distinguished between different viral genome sequences and provided single nucleotide-specific selection pressure to significantly change the proportions of viruses in a mixed viral pool. This finding indicates the utility of this tool for virus selection without the need for antibiotics or reporter genes, which could potentially save time compared to other methods used for screening and purifying mutant viruses.},
}

Animals
Bacterial Proteins/*genetics/metabolism
Base Sequence
*CRISPR-Cas Systems
Cercopithecus aethiops
Clustered Regularly Interspaced Short Palindromic Repeats
Deoxyribonuclease I/genetics/metabolism
Endonucleases/*genetics/metabolism
Gene Editing/*methods
Genes, Reporter
*Genome, Viral
Green Fluorescent Proteins/genetics/metabolism
HEK293 Cells
Herpesvirus 1, Human/*genetics/metabolism
Homologous Recombination
Humans
Lentivirus/genetics/metabolism
Plasmids/chemistry/metabolism
RNA, Guide/*genetics/metabolism
Selection, Genetic
Vero Cells
Virus Replication

@article {pmid30201707,
year = {2018},
author = {Yarrington, RM and Verma, S and Schwartz, S and Trautman, JK and Carroll, D},
title = {Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {},
number = {},
pages = {},
doi = {10.1073/pnas.1810062115},
pmid = {30201707},
issn = {1091-6490},
abstract = {Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.},
}

@article {pmid29621632,
year = {2018},
author = {Zhang, J and Song, F and Sun, Y and Yu, K and Xiang, J},
title = {CRISPR/Cas9-mediated deletion of EcMIH shortens metamorphosis time from mysis larva to postlarva of Exopalaemon carinicauda.},
journal = {Fish & shellfish immunology},
volume = {77},
number = {},
pages = {244-251},
doi = {10.1016/j.fsi.2018.04.002},
pmid = {29621632},
issn = {1095-9947},
mesh = {Amino Acid Sequence ; Animals ; Arthropod Proteins/chemistry/*genetics ; Base Sequence ; CRISPR-Cas Systems/*physiology ; Gene Expression Profiling ; Invertebrate Hormones/chemistry/*genetics ; Larva/genetics/growth & development ; Molting/*genetics ; Palaemonidae/*genetics/growth & development ; Phylogeny ; Sequence Alignment ; },
abstract = {The recently emerged CRISPR/Cas9 technology is the most flexible means to produce targeted mutations at the genomic loci in a variety of organisms. In Crustaceans, molt-inhibiting hormone (MIH) is an important negative-regulatory factor and plays a key role in suppressing the molting process. However, whether precise disruption of MIH in crustacean can be achieved and successfully used to improve the development and growth has not been proved. In this research, the complementary DNA (cDNA) and genomic DNA, including flanking regions of the MIH gene (EcMIH) of ridgetail white prawn Exopalaemon carinicauda, were cloned and sequenced. Sequence analysis revealed that EcMIH was composed of three exons and two introns. Analysis by RT-PCR showed that EcMIH mainly expressed in eyestalks. During different development periods, EcMIH was highest in juvenile stage and extremely low in others but adult prawns eyestalks. In addition, we applied CRISPR/Cas9 technology to generate EcMIH knock-out (KO) prawns and then analyzed the changes in their phenotypes. We efficiently generated 12 EcMIH-KO prawns out of 250 injected one-cell stage embryos and the mutant rate reached 4.8% after embryo injection with one sgRNA targeting the second exon of EcMIH. The EcMIH-KO prawns exhibited increased the body length and shortened the metamorphosis time of larvae from mysis larva to postlarva. Meanwhile, EcMIH-KO did not cause the health problems such as early stage death or deformity. In conclusion, we successfully obtained EcMIH gene and generated EcMIH-KO prawns using CRISPR/Cas9 technology. This study will certainly lead to a wide application prospect of MIH gene in prawns breeding.},
}

Amino Acid Sequence
Animals
Arthropod Proteins/chemistry/*genetics
Base Sequence
CRISPR-Cas Systems/*physiology
Gene Expression Profiling
Invertebrate Hormones/chemistry/*genetics
Larva/genetics/growth & development
Molting/*genetics
Palaemonidae/*genetics/growth & development
Phylogeny
Sequence Alignment

@article {pmid29555241,
year = {2018},
author = {Klaassen, H and Wang, Y and Adamski, K and Rohner, N and Kowalko, JE},
title = {CRISPR mutagenesis confirms the role of oca2 in melanin pigmentation in Astyanax mexicanus.},
journal = {Developmental biology},
volume = {441},
number = {2},
pages = {313-318},
doi = {10.1016/j.ydbio.2018.03.014},
pmid = {29555241},
issn = {1095-564X},
mesh = {Albinism/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Characiformes/*genetics/metabolism ; Fish Proteins/*genetics/metabolism ; *Gene Editing ; Melatonin/biosynthesis/*genetics ; Membrane Transport Proteins/*genetics/metabolism ; Pigmentation/*genetics ; },
abstract = {Understanding the genetic basis of trait evolution is critical to identifying the mechanisms that generated the immense amount of diversity observable in the living world. However, genetically manipulating organisms from natural populations with evolutionary adaptations remains a significant challenge. Astyanax mexicanus exists in two interfertile forms, a surface-dwelling form and multiple independently evolved cave-dwelling forms. Cavefish have evolved a number of morphological and behavioral traits and multiple quantitative trait loci (QTL) analyses have been performed to identify loci underlying these traits. These studies provide a unique opportunity to identify and test candidate genes for these cave-specific traits. We have leveraged the CRISPR/Cas9 genome editing techniques to characterize the effects of mutations in oculocutaneous albinism II (oca2), a candidate gene hypothesized to be responsible for the evolution of albinism in A. mexicanus cave populations. We generated oca2 mutant surface A. mexicanus. Surface fish with oca2 mutations are albino due to a disruption in the first step of the melanin synthesis pathway, the same step that is disrupted in albino cavefish. Hybrid offspring from crosses between oca2 mutant surface and cavefish are albino, definitively demonstrating the role of this gene in the evolution of albinism in this species. This research elucidates the role oca2 plays in pigmentation in fish, and establishes that this gene is solely responsible for the evolution of albinism in multiple cavefish populations. Finally, it demonstrates the utility of using genome editing to investigate the genetic basis of trait evolution.},
}

Albinism/genetics/metabolism
Animals
*CRISPR-Cas Systems
Characiformes/*genetics/metabolism
Fish Proteins/*genetics/metabolism
*Gene Editing
Melatonin/biosynthesis/*genetics
Membrane Transport Proteins/*genetics/metabolism
Pigmentation/*genetics

@article {pmid29555218,
year = {2018},
author = {Hoeksema, F and Karpilow, J and Luitjens, A and Lagerwerf, F and Havenga, M and Groothuizen, M and Gillissen, G and Lemckert, AAC and Jiang, B and Tripp, RA and Yallop, C},
title = {Enhancing viral vaccine production using engineered knockout vero cell lines - A second look.},
journal = {Vaccine},
volume = {36},
number = {16},
pages = {2093-2103},
doi = {10.1016/j.vaccine.2018.03.010},
pmid = {29555218},
issn = {1873-2518},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Batch Cell Culture Techniques ; CRISPR-Cas Systems ; Cercopithecus aethiops ; Gene Knockout Techniques ; Gene Targeting ; *Genetic Engineering ; Poliovirus/genetics/immunology ; Poliovirus Vaccines/chemistry/immunology ; Rotavirus/genetics/immunology ; Rotavirus Vaccines/genetics/immunology ; *Vero Cells ; *Viral Vaccines ; },
abstract = {The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications (van der Sanden et al., 2016; Wu et al., 2017) that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industry manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.},
}

Animals
*Batch Cell Culture Techniques
CRISPR-Cas Systems
Cercopithecus aethiops
Gene Knockout Techniques
Gene Targeting
*Genetic Engineering
Poliovirus/genetics/immunology
Poliovirus Vaccines/chemistry/immunology
Rotavirus/genetics/immunology
Rotavirus Vaccines/genetics/immunology
*Vero Cells
*Viral Vaccines

@article {pmid29146998,
year = {2017},
author = {Yablonovitch, AL and Fu, J and Li, K and Mahato, S and Kang, L and Rashkovetsky, E and Korol, AB and Tang, H and Michalak, P and Zelhof, AC and Nevo, E and Li, JB},
title = {Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1570},
doi = {10.1038/s41467-017-01658-2},
pmid = {29146998},
issn = {2041-1723},
support = {R01 GM124215/GM/NIGMS NIH HHS/United States ; R01 MH115080/MH/NIMH NIH HHS/United States ; T32 GM008294/GM/NIGMS NIH HHS/United States ; R21 EY024125/EY/NEI NIH HHS/United States ; R01 GM102484/GM/NIGMS NIH HHS/United States ; },
mesh = {AC133 Antigen/*genetics ; Adaptation, Physiological/*genetics ; Animals ; CRISPR-Cas Systems/genetics ; Drosophila melanogaster/*genetics ; Evolution, Molecular ; Female ; Gene Expression Profiling ; Gene Expression Regulation/*genetics ; Genome/genetics ; Glutathione Transferase/metabolism ; Microclimate ; Phosphoprotein Phosphatases/genetics ; Polymorphism, Single Nucleotide/genetics ; Principal Component Analysis ; RNA Editing/*genetics ; Temperature ; },
abstract = {Determining the mechanisms by which a species adapts to its environment is a key endeavor in the study of evolution. In particular, relatively little is known about how transcriptional processes are fine-tuned to adjust to different environmental conditions. Here we study Drosophila melanogaster from 'Evolution Canyon' in Israel, which consists of two opposing slopes with divergent microclimates. We identify several hundred differentially expressed genes and dozens of differentially edited sites between flies from each slope, correlate these changes with genetic differences, and use CRISPR mutagenesis to validate that an intronic SNP in prominin regulates its editing levels. We also demonstrate that while temperature affects editing levels at more sites than genetic differences, genetically regulated sites tend to be less affected by temperature. This work shows the extent to which gene expression and RNA editing differ between flies from different microclimates, and provides insights into the regulation responsible for these differences.},
}

AC133 Antigen/*genetics
Adaptation, Physiological/*genetics
Animals
CRISPR-Cas Systems/genetics
Drosophila melanogaster/*genetics
Evolution, Molecular
Female
Gene Expression Profiling
Gene Expression Regulation/*genetics
Genome/genetics
Glutathione Transferase/metabolism
Microclimate
Phosphoprotein Phosphatases/genetics
Polymorphism, Single Nucleotide/genetics
Principal Component Analysis
RNA Editing/*genetics
Temperature

@article {pmid29133799,
year = {2017},
author = {Saunderson, EA and Stepper, P and Gomm, JJ and Hoa, L and Morgan, A and Allen, MD and Jones, JL and Gribben, JG and Jurkowski, TP and Ficz, G},
title = {Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.},
journal = {Nature communications},
volume = {8},
number = {1},
pages = {1450},
doi = {10.1038/s41467-017-01078-2},
pmid = {29133799},
issn = {2041-1723},
support = {MR/M01892X/1//Medical Research Council/United Kingdom ; },
mesh = {Breast/*cytology/metabolism ; CRISPR-Cas Systems/genetics ; Cell Transformation, Neoplastic/*genetics ; Cells, Cultured ; Cellular Senescence/*genetics ; Cyclin-Dependent Kinase Inhibitor p16/metabolism ; Cyclin-Dependent Kinase Inhibitor p18/genetics ; DNA (Cytosine-5-)-Methyltransferases/genetics ; DNA Methylation/*genetics ; Epigenomics ; Female ; Gene Editing/*methods ; Humans ; Kruppel-Like Transcription Factors/genetics ; PTEN Phosphohydrolase/genetics ; Promoter Regions, Genetic/genetics ; Tumor Suppressor Proteins/genetics ; },
abstract = {Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.},
}

Breast/*cytology/metabolism
CRISPR-Cas Systems/genetics
Cell Transformation, Neoplastic/*genetics
Cells, Cultured
Cellular Senescence/*genetics
Cyclin-Dependent Kinase Inhibitor p16/metabolism
Cyclin-Dependent Kinase Inhibitor p18/genetics
DNA (Cytosine-5-)-Methyltransferases/genetics
DNA Methylation/*genetics
Epigenomics
Female
Gene Editing/*methods
Humans
Kruppel-Like Transcription Factors/genetics
PTEN Phosphohydrolase/genetics
Promoter Regions, Genetic/genetics
Tumor Suppressor Proteins/genetics

@article {pmid27558837,
year = {2016},
author = {Zhang, Y and Liang, Z and Zong, Y and Wang, Y and Liu, J and Chen, K and Qiu, JL and Gao, C},
title = {Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA.},
journal = {Nature communications},
volume = {7},
number = {},
pages = {12617},
doi = {10.1038/ncomms12617},
pmid = {27558837},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Editing/legislation & jurisprudence/*methods ; Genes, Plant/*genetics ; Homozygote ; Mutation ; Plants, Genetically Modified/*genetics ; Polyploidy ; RNA/genetics ; Triticum/*genetics ; },
abstract = {Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.},
}

CRISPR-Cas Systems/*genetics
DNA/genetics
Gene Editing/legislation & jurisprudence/*methods
Genes, Plant/*genetics
Homozygote
Mutation
Plants, Genetically Modified/*genetics
Polyploidy
RNA/genetics
Triticum/*genetics

@article {pmid27363581,
year = {2016},
author = {Nguyen, DP and Miyaoka, Y and Gilbert, LA and Mayerl, SJ and Lee, BH and Weissman, JS and Conklin, BR and Wells, JA},
title = {Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity.},
journal = {Nature communications},
volume = {7},
number = {},
pages = {12009},
doi = {10.1038/ncomms12009},
pmid = {27363581},
issn = {2041-1723},
support = {U01 CA168370/CA/NCI NIH HHS/United States ; U01 HL099997/HL/NHLBI NIH HHS/United States ; P50 GM102706/GM/NIGMS NIH HHS/United States ; R01 DA036858/DA/NIDA NIH HHS/United States ; R01 CA191018/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Bacterial Proteins/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/*metabolism ; Epigenesis, Genetic ; Estrogen Antagonists/pharmacology ; Gene Editing/*methods ; Gene Expression Regulation ; Glucocorticoids/pharmacology ; Plasmids ; Protein Binding ; *Protein Domains ; Receptors, Cytoplasmic and Nuclear/drug effects/*metabolism ; Receptors, Estrogen/drug effects/*metabolism ; Receptors, Glucocorticoid/drug effects/*metabolism ; Staphylococcus aureus ; Streptococcus pyogenes ; Tamoxifen/analogs & derivatives/pharmacology ; },
abstract = {Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.},
}

Bacterial Proteins/*metabolism
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Endonucleases/*metabolism
Epigenesis, Genetic
Estrogen Antagonists/pharmacology
Gene Editing/*methods
Gene Expression Regulation
Glucocorticoids/pharmacology
Plasmids
Protein Binding
*Protein Domains
Receptors, Cytoplasmic and Nuclear/drug effects/*metabolism
Receptors, Estrogen/drug effects/*metabolism
Receptors, Glucocorticoid/drug effects/*metabolism
Staphylococcus aureus
Streptococcus pyogenes
Tamoxifen/analogs & derivatives/pharmacology

@article {pmid27317309,
year = {2016},
author = {Li, Q and Zhang, D and Chen, M and Liang, W and Wei, J and Qi, Y and Yuan, Z},
title = {Development of japonica Photo-Sensitive Genic Male Sterile Rice Lines by Editing Carbon Starved Anther Using CRISPR/Cas9.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {43},
number = {6},
pages = {415-419},
doi = {10.1016/j.jgg.2016.04.011},
pmid = {27317309},
issn = {1673-8527},
mesh = {CRISPR-Cas Systems/*genetics ; Carbon/*metabolism ; *Gene Editing ; Genes, Plant/genetics ; Oryza/*genetics/metabolism/*radiation effects ; Plant Infertility/*genetics/*radiation effects ; },
}

CRISPR-Cas Systems/*genetics
Carbon/*metabolism
*Gene Editing
Genes, Plant/genetics
Oryza/*genetics/metabolism/*radiation effects
Plant Infertility/*genetics/*radiation effects

@article {pmid30198342,
year = {2018},
author = {Dangi, AK and Sharma, B and Hill, RT and Shukla, P},
title = {Bioremediation through microbes: systems biology and metabolic engineering approach.},
journal = {Critical reviews in biotechnology},
volume = {},
number = {},
pages = {1-20},
doi = {10.1080/07388551.2018.1500997},
pmid = {30198342},
issn = {1549-7801},
abstract = {Today, environmental pollution is a serious problem, and bioremediation can play an important role in cleaning contaminated sites. Remediation strategies, such as chemical and physical approaches, are not enough to mitigate pollution problems because of the continuous generation of novel recalcitrant pollutants due to anthropogenic activities. Bioremediation using microbes is an eco-friendly and socially acceptable alternative to conventional remediation approaches. Many microbes with a bioremediation potential have been isolated and characterized but, in many cases, cannot completely degrade the targeted pollutant or are ineffective in situations with mixed wastes. This review envisages advances in systems biology (SB), which enables the analysis of microbial behavior at a community level under different environmental stresses. By applying a SB approach, crucial preliminary information can be obtained for metabolic engineering (ME) of microbes for their enhanced bioremediation capabilities. This review also highlights the integrated SB and ME tools and techniques for bioremediation purposes.},
}

@article {pmid30096237,
year = {2018},
author = {Li, R and Zhang, L and Wang, L and Chen, L and Zhao, R and Sheng, J and Shen, L},
title = {Reduction of Tomato-Plant Chilling Tolerance by CRISPR-Cas9-Mediated SlCBF1 Mutagenesis.},
journal = {Journal of agricultural and food chemistry},
volume = {66},
number = {34},
pages = {9042-9051},
doi = {10.1021/acs.jafc.8b02177},
pmid = {30096237},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; Cold Temperature ; Gene Knockout Techniques ; Lycopersicon esculentum/genetics/*physiology ; Mutagenesis ; Plant Proteins/*genetics/metabolism ; *Stress, Physiological ; },
abstract = {Chilling stress is the main constraint in tomato (Solanum lycopersicum) production, as this is a chilling-sensitive horticultural crop. The highly conserved C-repeat binding factors (CBFs) are cold-response-system components found in many species. In this study, we generated slcbf1 mutants using the CRISPR-Cas9 system and investigated the role of SlCBF1 in tomato-plant chilling tolerances. The slcbf1 mutants exhibited more severe chilling-injury symptoms with higher electrolyte leakage and malondialdehyde levels than wild-type (WT) plants. Additionally, slcbf1 mutants showed lower proline and protein contents and higher hydrogen peroxide contents and activities of antioxidant enzymes than WT plants. Knockout of SlCBF1 significantly increased indole acetic acid contents but decreased methyl jasmonate, abscisic acid, and zeatin riboside contents. The reduced chilling tolerance of the slcbf1 mutants was further reflected by the down-regulation of CBF-related genes. These results contribute to a better understanding of the molecular basis underlying SlCBF1 mediation of tomato chilling sensitivity.},
}

CRISPR-Cas Systems
Cold Temperature
Gene Knockout Techniques
Lycopersicon esculentum/genetics/*physiology
Mutagenesis
Plant Proteins/*genetics/metabolism
*Stress, Physiological

@article {pmid29676032,
year = {2018},
author = {Acosta, S and Fiore, L and Carota, IA and Oliver, G},
title = {Use of two gRNAs for CRISPR/Cas9 improves bi-allelic homologous recombination efficiency in mouse embryonic stem cells.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {56},
number = {5},
pages = {e23212},
doi = {10.1002/dvg.23212},
pmid = {29676032},
issn = {1526-968X},
support = {R01 EY012162/EY/NEI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics/physiology ; Gene Editing/*methods ; Gene Targeting/methods ; Genetic Engineering/methods ; Homologous Recombination/genetics ; Mice/embryology ; Mouse Embryonic Stem Cells/metabolism ; RNA, Guide/*genetics/metabolism ; Regulatory Sequences, Nucleic Acid/genetics ; },
abstract = {Targeted genome editing in mouse embryonic stem cells (ESCs) is a powerful resource to functionally characterize genes and regulatory elements. The use of the CRISPR/Cas9 genome editing approach has remarkably improved the time and efficiency of targeted recombination. However, the efficiency of this protocol is still far from ideal when aiming for bi-allelic homologous recombination, requiring at least two independent targeting recombination events. Here we describe an improved protocol that uses two gRNAs flanking the selected targeted region, leading to highly efficient homologous recombination in mouse ESCs. The bi-allelic recombination targeting efficiency is over 90% when using two gRNAs together with the inhibition of non-homologous end-joint repair. Moreover, this technique is compatible with the generation of knocked-in mice and the use of ESC-derived differentiation protocols, therefore facilitating and accelerating the gene targeting in mice and ESCs.},
}

Alleles
Animals
CRISPR-Cas Systems/genetics/physiology
Gene Editing/*methods
Gene Targeting/methods
Genetic Engineering/methods
Homologous Recombination/genetics
Mice/embryology
Mouse Embryonic Stem Cells/metabolism
RNA, Guide/*genetics/metabolism
Regulatory Sequences, Nucleic Acid/genetics

@article {pmid29637228,
year = {2018},
author = {Wu, H and Liu, Q and Shi, H and Xie, J and Zhang, Q and Ouyang, Z and Li, N and Yang, Y and Liu, Z and Zhao, Y and Lai, C and Ruan, D and Peng, J and Ge, W and Chen, F and Fan, N and Jin, Q and Liang, Y and Lan, T and Yang, X and Wang, X and Lei, Z and Doevendans, PA and Sluijter, JPG and Wang, K and Li, X and Lai, L},
title = {Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {75},
number = {19},
pages = {3593-3607},
doi = {10.1007/s00018-018-2810-3},
pmid = {29637228},
issn = {1420-9071},
support = {81702115//National Natural Science Foundation of China/ ; 81672317//National Natural Science Foundation of China/ ; 2017YFA0105103//National Key R&D Program of China/ ; },
mesh = {Animals ; Animals, Genetically Modified ; Animals, Newborn ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Cloning, Molecular/*methods ; Cloning, Organism/*methods ; Embryo, Mammalian ; Endonucleases/*genetics/metabolism ; Female ; Fetus ; Gene Editing/*methods ; Genome/genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Male ; Mammals/embryology/genetics ; Mutagenesis ; Nuclear Transfer Techniques ; Pregnancy ; RNA, Guide/genetics ; RNA, Transfer/*genetics ; Rabbits ; Swine ; Swine, Miniature ; },
abstract = {CRISPR/Cpf1 features a number of properties that are distinct from CRISPR/Cas9 and provides an excellent alternative to Cas9 for genome editing. To date, genome engineering by CRISPR/Cpf1 has been reported only in human cells and mouse embryos of mammalian systems and its efficiency is ultimately lower than that of Cas9 proteins from Streptococcus pyogenes. The application of CRISPR/Cpf1 for targeted mutagenesis in other animal models has not been successfully verified. In this study, we designed and optimized a guide RNA (gRNA) transcription system by inserting a transfer RNA precursor (pre-tRNA) sequence downstream of the gRNA for Cpf1, protecting gRNA from immediate digestion by 3'-to-5' exonucleases. Using this new gRNAtRNA system, genome editing, including indels, large fragment deletion and precise point mutation, was induced in mammalian systems, showing significantly higher efficiency than the original Cpf1-gRNA system. With this system, gene-modified rabbits and pigs were generated by embryo injection or somatic cell nuclear transfer (SCNT) with an efficiency comparable to that of the Cas9 gRNA system. These results demonstrated that this refined gRNAtRNA system can boost the targeting capability of CRISPR/Cpf1 toolkits.},
}

Animals
Animals, Genetically Modified
Animals, Newborn
Bacterial Proteins/*genetics/metabolism
CRISPR-Cas Systems/*genetics
Cells, Cultured
Cloning, Molecular/*methods
Cloning, Organism/*methods
Embryo, Mammalian
Endonucleases/*genetics/metabolism
Female
Fetus
Gene Editing/*methods
Genome/genetics
HEK293 Cells
HeLa Cells
Humans
Male
Mammals/embryology/genetics
Mutagenesis
Nuclear Transfer Techniques
Pregnancy
RNA, Guide/genetics
RNA, Transfer/*genetics
Rabbits
Swine
Swine, Miniature

@article {pmid29617297,
year = {2018},
author = {Watanabe, S and Sakurai, T and Nakamura, S and Miyoshi, K and Sato, M},
title = {The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones.},
journal = {International journal of molecular sciences},
volume = {19},
number = {4},
pages = {},
doi = {10.3390/ijms19041075},
pmid = {29617297},
issn = {1422-0067},
mesh = {Alleles ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; *Gene Targeting ; Genetic Vectors ; Genotype ; Glycoside Hydrolases/genetics ; Humans ; INDEL Mutation ; Mammals/genetics ; RNA, Guide ; Toxins, Biological/*genetics ; },
abstract = {Recent advances in genome editing systems such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) have facilitated genomic modification in mammalian cells. However, most systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. Here, we developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells. Cells were transfected with three expression vectors, each of which carries a guide RNA (gRNA), humanized Cas9 (hCas9) gene, or Clostridium perfringens-derived endo-&beta;-galactosidase C (EndoGalC) gene. Once EndoGalC is expressed in a cell, it digests the cell-surface &alpha;-Gal epitope, which is specifically recognized by BS-I-B₄ lectin (IB4). Three days after transfection, these cells were treated with cytotoxin saporin-conjugated IB4 (IB4SAP) for 30 min at 37 &deg;C prior to cultivation in a normal medium. Untransfected cells and those weakly expressing EndoGalC will die due to the internalization of saporin. Cells transiently expressing EndoGalC strongly survive, and some of these surviving clones are expected to be genome-edited bi-allelic knockout (KO) clones due to their strong co-expression of gRNA and hCas9. When porcine &alpha;-1,3-galactosyltransferase gene, which can synthesize the &alpha;-Gal epitope, was attempted to be knocked out, 16.7% and 36.7% of the surviving clones were bi-allelic and mono-allelic knockout (KO) cells, respectively, which was in contrast to the isolation of clones in the absence of IB4SAP treatment. Namely, 0% and 13.3% of the resulting clones were bi-allelic and mono-allelic KO cells, respectively. A similar tendency was seen when other target genes such as DiGeorge syndrome critical region gene 2 and transforming growth factor-&beta; receptor type 1 gene were targeted to be knocked out. Our results indicate that a combination of the CRISPR/Cas9 system and targeted toxin technology using IB4SAP allows efficient enrichment of genome-edited clones, particularly bi-allelic KO clones.},
}

Alleles
Animals
Base Sequence
*CRISPR-Cas Systems
Gene Editing
Gene Knockout Techniques
*Gene Targeting
Genetic Vectors
Genotype
Glycoside Hydrolases/genetics
Humans
INDEL Mutation
Mammals/genetics
RNA, Guide
Toxins, Biological/*genetics

@article {pmid29614837,
year = {2018},
author = {Gao, J and Zhang, T and Xu, B and Jia, L and Xiao, B and Liu, H and Liu, L and Yan, H and Xia, Q},
title = {CRISPR/Cas9-Mediated Mutagenesis of Carotenoid Cleavage Dioxygenase 8 (CCD8) in Tobacco Affects Shoot and Root Architecture.},
journal = {International journal of molecular sciences},
volume = {19},
number = {4},
pages = {},
doi = {10.3390/ijms19041062},
pmid = {29614837},
issn = {1422-0067},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Dioxygenases/genetics/*metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/*metabolism ; Plant Roots/*enzymology/genetics/*metabolism ; Plant Shoots/*enzymology/genetics/*metabolism ; Tobacco/*enzymology/genetics/*metabolism ; },
abstract = {Strigolactones (SLs) are a class of phytohormones that regulate plant architecture. Carotenoid cleavage dioxygenase (CCD) genes are involved in the biosynthesis of SLs and are identified and characterized in many plants. However, the function of CCD genes in tobacco remains poorly understood. In this study, two closely related genes NtCCD8A and NtCCD8B were cloned from tobacco (Nicotiana tabacum L.). The two NtCCD8 genes are orthologues of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase 8 (SlCCD8) gene. NtCCD8A and NtCCD8B were primarily expressed in tobacco roots, but low expression levels of these genes were detected in all plant tissues, and their transcript levels significantly increased in response to phosphate limitation. NtCCD8A and NtCCD8B mutations were introduced into tobacco using the CRISPR/Cas9 system and transgenic tobacco lines for both ntccd8 mutant alleles were identified. The ntccd8a and ntccd8b mutant alleles were inactivated by a deletion of three nucleotides and insertion of one nucleotide, respectively, both of which led to the production of premature stop codons. The ntccd8 mutants had increased shoot branching, reduced plant height, increased number of leaves and nodes, and reduced total plant biomass compared to wild-type plants; however, the root-to-shoot ratio was unchanged. In addition, mutant lines had shorter primary roots and more of lateral roots than wild type. These results suggest that NtCCD8 genes are important for changes in tobacco plant architecture.},
}

CRISPR-Cas Systems/genetics/physiology
Dioxygenases/genetics/*metabolism
Gene Expression Regulation, Plant
Plant Proteins/genetics/*metabolism
Plant Roots/*enzymology/genetics/*metabolism
Plant Shoots/*enzymology/genetics/*metabolism
Tobacco/*enzymology/genetics/*metabolism

@article {pmid29596855,
year = {2018},
author = {Vad-Nielsen, J and Nielsen, AL and Luo, Y},
title = {Simple method for assembly of CRISPR synergistic activation mediator gRNA expression array.},
journal = {Journal of biotechnology},
volume = {274},
number = {},
pages = {54-57},
doi = {10.1016/j.jbiotec.2018.03.018},
pmid = {29596855},
issn = {1873-4863},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; *Gene Regulatory Networks ; Humans ; Neoplasms/genetics ; Oligonucleotide Array Sequence Analysis/*methods ; RNA, Guide/*genetics ; },
abstract = {When studying complex interconnected regulatory networks, effective methods for simultaneously manipulating multiple genes expression are paramount. Previously, we have developed a simple method for generation of an all-in-one CRISPR gRNA expression array. We here present a Golden Gate Assembly-based system of synergistic activation mediator (SAM) compatible CRISPR/dCas9 gRNA expression array for the simultaneous activation of multiple genes. Using this system, we demonstrated the simultaneous activation of the transcription factors, TWIST, SNAIL, SLUG, and ZEB1 a human breast cancer cell line.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Gene Expression Profiling/*methods
Gene Expression Regulation, Neoplastic
*Gene Regulatory Networks
Humans
Neoplasms/genetics
Oligonucleotide Array Sequence Analysis/*methods
RNA, Guide/*genetics

@article {pmid29247600,
year = {2018},
author = {Xie, H and Tang, L and He, X and Liu, X and Zhou, C and Liu, J and Ge, X and Li, J and Liu, C and Zhao, J and Qu, J and Song, Z and Gu, F},
title = {SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.},
journal = {Biotechnology journal},
volume = {13},
number = {4},
pages = {e1700561},
doi = {10.1002/biot.201700561},
pmid = {29247600},
issn = {1860-7314},
mesh = {Bacterial Proteins/metabolism ; CRISPR-Cas Systems ; *DNA, Intergenic ; Endonucleases/*metabolism ; Francisella/*enzymology ; Genetic Engineering ; HEK293 Cells ; Humans ; RNA, Guide ; Staphylococcus aureus/*enzymology ; Streptococcus pyogenes/*enzymology ; Substrate Specificity ; },
abstract = {CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.},
}

Bacterial Proteins/metabolism
CRISPR-Cas Systems
*DNA, Intergenic
Endonucleases/*metabolism
Francisella/*enzymology
Genetic Engineering
HEK293 Cells
Humans
RNA, Guide
Staphylococcus aureus/*enzymology
Streptococcus pyogenes/*enzymology
Substrate Specificity

@article {pmid29124278,
year = {2018},
author = {Merkert, S and Martin, U},
title = {Targeted Gene Editing in Human Pluripotent Stem Cells Using Site-Specific Nucleases.},
journal = {Advances in biochemical engineering/biotechnology},
volume = {163},
number = {},
pages = {169-186},
doi = {10.1007/10_2017_25},
pmid = {29124278},
issn = {0724-6145},
mesh = {*CRISPR-Cas Systems ; Endonucleases/*chemistry ; Gene Editing/*methods ; Humans ; Pluripotent Stem Cells/cytology/*metabolism ; },
abstract = {Introduction of induced pluripotent stem cell (iPSC) technology and site-directed nucleases brought a major breakthrough in the development of regenerative therapies and biomedical research. With the advancement of ZFNs, TALENs, and the CRISPR/Cas9 technology, straightforward and precise manipulation of the genome of human pluripotent stem cells (PSC) became possible, allowing relatively easy and fast generation of gene knockouts, integration of transgenes, or even introduction of single nucleotide changes for correction or introduction of disease-specific mutations. We review current applications of site-specific nucleases in human PSCs and focus on trends and challenges for efficient gene editing and improvement of targeting strategies. Graphical Abstract.},
}

*CRISPR-Cas Systems
Endonucleases/*chemistry
Gene Editing/*methods
Humans
Pluripotent Stem Cells/cytology/*metabolism

@article {pmid27572891,
year = {2016},
author = {Pankowicz, FP and Barzi, M and Legras, X and Hubert, L and Mi, T and Tomolonis, JA and Ravishankar, M and Sun, Q and Yang, D and Borowiak, M and Sumazin, P and Elsea, SH and Bissig-Choisat, B and Bissig, KD},
title = {Reprogramming metabolic pathways in vivo with CRISPR/Cas9 genome editing to treat hereditary tyrosinaemia.},
journal = {Nature communications},
volume = {7},
number = {},
pages = {12642},
doi = {10.1038/ncomms12642},
pmid = {27572891},
issn = {2041-1723},
support = {P30 CA125123/CA/NCI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; R01 HL134510/HL/NHLBI NIH HHS/United States ; T32 HL092332/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Proliferation/genetics ; Cyclohexanones/therapeutic use ; Disease Models, Animal ; Enzyme Inhibitors/therapeutic use ; Exons/genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hepatocytes/metabolism ; Humans ; Hydrolases/genetics ; Liver/cytology/pathology ; Metabolic Networks and Pathways/*genetics ; Mice ; Mice, Inbred Strains ; Mice, Knockout ; Nitrobenzoates/therapeutic use ; Oxidoreductases/antagonists & inhibitors/genetics/metabolism ; Phenotype ; Tyrosinemias/*genetics/metabolism/pathology/therapy ; },
abstract = {Many metabolic liver disorders are refractory to drug therapy and require orthotopic liver transplantation. Here we demonstrate a new strategy, which we call metabolic pathway reprogramming, to treat hereditary tyrosinaemia type I in mice; rather than edit the disease-causing gene, we delete a gene in a disease-associated pathway to render the phenotype benign. Using CRISPR/Cas9 in vivo, we convert hepatocytes from tyrosinaemia type I into the benign tyrosinaemia type III by deleting Hpd (hydroxyphenylpyruvate dioxigenase). Edited hepatocytes (Fah(-/-)/Hpd(-/-)) display a growth advantage over non-edited hepatocytes (Fah(-/-)/Hpd(+/+)) and, in some mice, almost completely replace them within 8 weeks. Hpd excision successfully reroutes tyrosine catabolism, leaving treated mice healthy and asymptomatic. Metabolic pathway reprogramming sidesteps potential difficulties associated with editing a critical disease-causing gene and can be explored as an option for treating other diseases.},
}

Animals
CRISPR-Cas Systems/*genetics
Cell Line
Cell Proliferation/genetics
Cyclohexanones/therapeutic use
Disease Models, Animal
Enzyme Inhibitors/therapeutic use
Exons/genetics
Gene Editing/*methods
Genetic Therapy/*methods
Hepatocytes/metabolism
Humans
Hydrolases/genetics
Liver/cytology/pathology
Metabolic Networks and Pathways/*genetics
Mice
Mice, Inbred Strains
Mice, Knockout
Nitrobenzoates/therapeutic use
Oxidoreductases/antagonists & inhibitors/genetics/metabolism
Phenotype
Tyrosinemias/*genetics/metabolism/pathology/therapy

@article {pmid30195930,
year = {2018},
author = {St John, E and Liu, Y and Podar, M and Stott, MB and Meneghin, J and Chen, Z and Lagutin, K and Mitchell, K and Reysenbach, AL},
title = {A new symbiotic nanoarchaeote (Candidatus Nanoclepta minutus) and its host (Zestosphaera tikiterensis gen. nov., sp. nov.) from a New Zealand hot spring.},
journal = {Systematic and applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.syapm.2018.08.005},
pmid = {30195930},
issn = {1618-0984},
abstract = {Three thermophilic Nanoarchaeota-Crenarchaeota symbiotic systems have been described. We obtained another stable anaerobic enrichment culture at 80°C, pH 6.0 from a New Zealand hot spring. The nanoarchaeote (Ncl-1) and its host (NZ3T) were isolated in co-culture and their genomes assembled. The small (∼200nm) flagellated cocci were often attached to larger cocci. Based on 16S rRNA gene similarity (88.4%) and average amino acid identity (52%), Ncl-1 is closely related to Candidatus Nanopusillus acidilobi. Their genomes both encode for archaeal flagella and partial glycolysis and gluconeogenesis pathways, but lack ATP synthase genes. Like Nanoarchaeum equitans, Ncl-1 has a CRISPR-Cas system. Ncl-1 also relies on its crenarchaeotal host for most of its biosynthetic needs. The host NZ3T was isolated and grows on proteinaceous substrates but not on sugars, alcohols, or fatty acids. NZ3T requires thiosulfate and grows best at 82°C, pH 6.0. NZ3T is most closely related to the Desulfurococcaceae, Ignisphaera aggregans (∼92% 16S rRNA gene sequence similarity, 45% AAI). Based on phylogenetic, physiological and genomic data, Ncl-1 and NZ3T represent novel genera in the Nanoarchaeota and the Desulfurococcaceae, respectively, with the proposed names Candidatus Nanoclepta minutus and Zestosphaera tikiterensis gen. nov., sp. nov., type strain NZ3T (=DSMZ 107634T=OCM 1213T).},
}

@article {pmid30195753,
year = {2018},
author = {Gao, Z and Herrera-Carrillo, E and Berkhout, B},
title = {RNA Polymerase II Activity of Type 3 Pol III Promoters.},
journal = {Molecular therapy. Nucleic acids},
volume = {12},
number = {},
pages = {135-145},
doi = {10.1016/j.omtn.2018.05.001},
pmid = {30195753},
issn = {2162-2531},
abstract = {In eukaryotes, three RNA polymerases (Pol I, II, and III) are responsible for the transcription of distinct subsets of genes. Gene-external type 3 Pol III promoters use defined transcription start and termination sites, and they are, therefore, widely used for small RNA expression, including short hairpin RNAs in RNAi applications and guide RNAs in CRISPR-Cas systems. We report that all three commonly used human Pol III promoters (7SK, U6, and H1) mediate luciferase reporter gene expression, which indicates Pol II activity, but to a different extent (H1 ≫ U6 > 7SK). We demonstrate that these promoters can recruit Pol II for transcribing extended messenger transcripts. Intriguingly, selective inhibition of Pol II stimulates the Pol III activity and vice versa, suggesting that two polymerase complexes compete for promoter usage. Pol II initiates transcription at the regular Pol III start site on the 7SK and U6 promoters, but Pol II transcription on the most active H1 promoter starts 8 nt upstream of the Pol III start site. This study provides functional evidence for the close relationship of Pol II and Pol III transcription. These mechanistic insights are important for optimal use of Pol III promoters, and they offer additional flexibility for biotechnology applications of these genetic elements.},
}

@article {pmid30193985,
year = {2018},
author = {Faure, G and Makarova, KS and Koonin, EV},
title = {CRISPR-Cas: Complex functional networks and multiple roles beyond adaptive immunity.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jmb.2018.08.030},
pmid = {30193985},
issn = {1089-8638},
abstract = {CRISPR-Cas is a prokaryotic adaptive immune system that functions by incorporating fragments of foreign DNA into CRISPR arrays. The arrays containing spacers derived from foreign DNA are transcribed, and the transcripts are processed to generate spacer-containing mature CRISPR-RNAs that are employed as guides to specifically recognize and cleave the DNA or RNA of the cognate parasitic genetic elements. The CRISPR-Cas systems show remarkable complexity and diversity of molecular organization and appear to be involved in various cellular functions that are distinct from, even if connected to, adaptive immunity. In this review, we discuss some of such functional links of CRISPR-Cas systems including their effect on horizontal gene transfer that can be either inhibitory or stimulatory, connections between CRISPR-Cas and DNA repair systems as well as programmed cell death and signal transduction mechanisms, and potential role of CRISPR-Cas in transposon integration and plasmid maintenance. The interplay between the primary function of CRISPR-Cas as an adaptive immunity mechanism and these other roles defines the richness of the biological effects of these systems and affects their spread among bacteria and archaea.},
}

@article {pmid30193161,
year = {2018},
author = {Vogel, P and Stafforst, T},
title = {Critical review on engineering deaminases for site-directed RNA editing.},
journal = {Current opinion in biotechnology},
volume = {55},
number = {},
pages = {74-80},
doi = {10.1016/j.copbio.2018.08.006},
pmid = {30193161},
issn = {1879-0429},
abstract = {The game-changing role of CRISPR/Cas for genome editing draw interest to programmable RNA-guided tools in general. Currently, we see a wave of papers pioneering the CRISPR/Cas system for RNA targeting, and applying them for site-directed RNA editing. Here, we exemplarily compare three recent RNA editing strategies that rely on three distinct RNA targeting mechanisms. We conclude that the CRISPR/Cas system seems not generally superior to other RNA targeting strategies in solving the most pressing problem in the RNA editing field, which is to obtain high efficiency in combination with high specificity. However, once achieved, RNA editing promises to complement or even outcompete DNA editing approaches in therapy, and also in some fields of basic research.},
}

@article {pmid30190322,
year = {2018},
author = {Shen, Y and Cohen, JL and Nicoloro, SM and Kelly, M and Yenilmez, B and Henriques, F and Tsagkaraki, E and Edwards, YJK and Hu, X and Friedline, RH and Kim, JK and Czech, MP},
title = {CRISPR delivery particles targeting nuclear receptor-interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA118.004554},
pmid = {30190322},
issn = {1083-351X},
abstract = {RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide (sg)RNA could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a "browning" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed "CRISPR-delivery particles," denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary preadipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte "browning" with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease.},
}

@article {pmid30190308,
year = {2018},
author = {Marino, ND and Zhang, JY and Borges, AL and Sousa, AA and Leon, LM and Rauch, BJ and Walton, RT and Berry, JD and Joung, JK and Kleinstiver, BP and Bondy-Denomy, J},
title = {Discovery of widespread Type I and Type V CRISPR-Cas inhibitors.},
journal = {Science (New York, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1126/science.aau5174},
pmid = {30190308},
issn = {1095-9203},
abstract = {Bacterial CRISPR-Cas systems protect their host from bacteriophages and other mobile genetic elements. Mobile elements, in turn, encode various anti-CRISPR (Acr) proteins to inhibit the immune function of CRISPR-Cas. To date, Acr proteins have been discovered for type I (subtypes I-D, I-E, and I-F) and type II (II-A and II-C) but not other CRISPR systems. Here we report the discovery of 12 acr genes, including inhibitors of type V-A and I-C CRISPR systems. Notably, AcrVA1 inhibits a broad spectrum of Cas12a (Cpf1) orthologs including MbCas12a, Mb3Cas12a, AsCas12a, and LbCas12a when assayed in human cells. The acr genes reported here provide useful biotechnological tools and mark the discovery of acr loci in many bacteria and phages.},
}

@article {pmid29891675,
year = {2018},
author = {Ishii, T and Hayakawa, H and Igawa, T and Sekiguchi, T and Sekiguchi, M},
title = {Specific binding of PCBP1 to heavily oxidized RNA to induce cell death.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {26},
pages = {6715-6720},
doi = {10.1073/pnas.1806912115},
pmid = {29891675},
issn = {1091-6490},
mesh = {Amino Acid Substitution ; Apoptosis/*physiology ; CRISPR-Cas Systems ; Caspase 3/physiology ; Conserved Sequence ; Guanine/analogs & derivatives/metabolism ; HeLa Cells ; Heterogeneous-Nuclear Ribonucleoprotein D/metabolism ; Heterogeneous-Nuclear Ribonucleoproteins/chemistry/*metabolism ; Humans ; Hydrogen Peroxide/pharmacology ; Oxidation-Reduction ; Protein Domains ; RNA, Messenger/chemistry/*metabolism ; Recombinant Proteins/chemistry/metabolism ; Structure-Activity Relationship ; },
abstract = {In aerobically growing cells, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine (8-oxoG), which induces alteration in their gene expression. We previously demonstrated that the human AUF1 protein binds to 8-oxoG in RNA to induce the selective degradation of oxidized messenger RNA. We herein report that the poly(C)-binding protein PCBP1 binds to more severely oxidized RNA to activate apoptosis-related reactions. While AUF1 binds to oligoribonucleotides carrying a single 8-oxoG, PCBP1 does not bind to such oligoribonucleotides but instead binds firmly to oligoribonucleotides in which two 8-oxoG residues are located nearby. PCBP1-deficient cells, constructed from the human HeLa S3 line using the CRISPR-Cas9 system, exhibited higher survival rates than HeLa S3 cells when small doses of hydrogen peroxide were applied. The levels of caspase-3 activation and PARP-1 cleavage in the PCBP1-deficient cells were significantly lower than those in wild-type cells. The structure-function relationship of PCBP1 was established with the use of PCBP1 mutant proteins in which the conserved KH domains were defective. Human cells appear to possess two distinct mechanisms, one controlled by AUF1 and the other by PCBP1, with the former functioning when messenger RNA is moderately oxidized and the latter operating when the RNA is more severely damaged.},
}

Amino Acid Substitution
Apoptosis/*physiology
CRISPR-Cas Systems
Caspase 3/physiology
Conserved Sequence
Guanine/analogs & derivatives/metabolism
HeLa Cells
Heterogeneous-Nuclear Ribonucleoprotein D/metabolism
Heterogeneous-Nuclear Ribonucleoproteins/chemistry/*metabolism
Humans
Hydrogen Peroxide/pharmacology
Oxidation-Reduction
Protein Domains
RNA, Messenger/chemistry/*metabolism
Recombinant Proteins/chemistry/metabolism
Structure-Activity Relationship

@article {pmid29730672,
year = {2018},
author = {Kocak, DD and Gersbach, CA},
title = {From CRISPR scissors to virus sensors.},
journal = {Nature},
volume = {557},
number = {7704},
pages = {168-169},
doi = {10.1038/d41586-018-04975-8},
pmid = {29730672},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Viruses ; *Gene Editing ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA Viruses
*Gene Editing

@article {pmid29531059,
year = {2018},
author = {Lapinaite, A and Doudna, JA and Cate, JHD},
title = {Programmable RNA recognition using a CRISPR-associated Argonaute.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {13},
pages = {3368-3373},
doi = {10.1073/pnas.1717725115},
pmid = {29531059},
issn = {1091-6490},
support = {R01 GM065050/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Argonaute Proteins/*chemistry/genetics/metabolism ; Bacteria/*genetics ; *CRISPR-Cas Systems ; Models, Biological ; RNA, Bacterial/*chemistry/genetics/metabolism ; RNA, Guide/*chemistry/genetics/metabolism ; },
abstract = {Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5'-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5' nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5'-BrdU-modified gRNAs, we mapped the seed region of the gRNA and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the sixth and seventh positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific adenosine-to-inosine-edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5'-BrdU-modified gRNAs can be used as a highly specific RNA-targeting platform to probe RNA biology.},
}

Argonaute Proteins/*chemistry/genetics/metabolism
Bacteria/*genetics
*CRISPR-Cas Systems
Models, Biological
RNA, Bacterial/*chemistry/genetics/metabolism
RNA, Guide/*chemistry/genetics/metabolism

@article {pmid28119373,
year = {2017},
author = {Liu, ET and Bolcun-Filas, E and Grass, DS and Lutz, C and Murray, S and Shultz, L and Rosenthal, N},
title = {Of mice and CRISPR: The post-CRISPR future of the mouse as a model system for the human condition.},
journal = {EMBO reports},
volume = {18},
number = {2},
pages = {187-193},
doi = {10.15252/embr.201643717},
pmid = {28119373},
issn = {1469-3178},
support = {U42 OD010921/OD/NIH HHS/United States ; U54 OD020351/OD/NIH HHS/United States ; UM1 OD023222/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Genetic Engineering/methods/*trends ; Humans ; Mice/*genetics ; },
}

Animals
*CRISPR-Cas Systems
*Disease Models, Animal
Genetic Engineering/methods/*trends
Humans
Mice/*genetics

@article {pmid27898061,
year = {2016},
author = {Kuscu, C and Adli, M},
title = {CRISPR-Cas9-AID base editor is a powerful gain-of-function screening tool.},
journal = {Nature methods},
volume = {13},
number = {12},
pages = {983-984},
doi = {10.1038/nmeth.4076},
pmid = {27898061},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; *Gain of Function Mutation ; *Gene Editing ; High-Throughput Screening Assays/*methods ; Humans ; },
}

*CRISPR-Cas Systems
*Gain of Function Mutation
*Gene Editing
High-Throughput Screening Assays/*methods
Humans

@article {pmid30189348,
year = {2018},
author = {Verkuijl, SA and Rots, MG},
title = {The influence of eukaryotic chromatin state on CRISPR-Cas9 editing efficiencies.},
journal = {Current opinion in biotechnology},
volume = {55},
number = {},
pages = {68-73},
doi = {10.1016/j.copbio.2018.07.005},
pmid = {30189348},
issn = {1879-0429},
abstract = {CRISPR/Cas technologies have rapidly become in routine use for site-directed genetic or transcriptional manipulation. Despite this, the efficiency of CRISPR/Cas9 functioning cannot entirely be predicted, and it is not fully understood which factors contribute to this variability. Recent studies indicate that heterochromatin can negatively affect Cas9 binding and functioning. Investigating chromatin factors indicates that DNA cytosine-5 methylation does not directly block Cas9 binding. Nucleosomes, however, can completely block Cas9 access to DNA in cell-free assays and present a substantial hurdle in vivo. In addition to being associated with an open chromatin state, active transcription can directly stimulate DNA cleavage by influencing Cas9 release rates in a strand-specific manner. With these insights and a better understanding of genome-wide chromatin and transcription states, CRISPR/Cas9 effectiveness and reliability can be improved.},
}

@article {pmid30189098,
year = {2018},
author = {Radovcic, M and Killelea, T and Savitskaya, E and Wettstein, L and Bolt, EL and Ivancic-Bace, I},
title = {CRISPR-Cas adaptation in Escherichia coli requires RecBCD helicase but not nuclease activity, is independent of homologous recombination, and is antagonized by 5' ssDNA exonucleases.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gky799},
pmid = {30189098},
issn = {1362-4962},
abstract = {Prokaryotic adaptive immunity is established against mobile genetic elements (MGEs) by 'naïve adaptation' when DNA fragments from a newly encountered MGE are integrated into CRISPR-Cas systems. In Escherichia coli, DNA integration catalyzed by Cas1-Cas2 integrase is well understood in mechanistic and structural detail but much less is known about events prior to integration that generate DNA for capture by Cas1-Cas2. Naïve adaptation in E. coli is thought to depend on the DNA helicase-nuclease RecBCD for generating DNA fragments for capture by Cas1-Cas2. The genetics presented here show that naïve adaptation does not require RecBCD nuclease activity but that helicase activity may be important. RecA loading by RecBCD inhibits adaptation explaining previously observed adaptation phenotypes that implicated RecBCD nuclease activity. Genetic analysis of other E. coli nucleases and naïve adaptation revealed that 5' ssDNA tailed DNA molecules promote new spacer acquisition. We show that purified E. coli Cas1-Cas2 complex binds to and nicks 5' ssDNA tailed duplexes and propose that E. coli Cas1-Cas2 nuclease activity on such DNA structures supports naïve adaptation.},
}

@article {pmid30183125,
year = {2018},
author = {Ortigosa, A and Gimenez-Ibanez, S and Leonhardt, N and Solano, R},
title = {Design of a bacterial speck resistant tomato by CRISPR/Cas9-mediated editing of SlJAZ2.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13006},
pmid = {30183125},
issn = {1467-7652},
abstract = {Due to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade-off is a major challenge for obtaining broad-spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co-receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by Pto DC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co-receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C-terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to Pto DC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in SljazΔjas plants. Our results solve the defense trade-off in a crop, by spatially uncoupling the SA-JA hormonal antagonism at the stomata, entry gates of specific microbes such as Pto DC3000. Moreover, our results also constitute a novel CRISPR/Cas-based strategy for crop protection that could be readily implemented in the field. This article is protected by copyright. All rights reserved.},
}

@article {pmid29980564,
year = {2018},
author = {York, JR and Yuan, T and Lakiza, O and McCauley, DW},
title = {An ancestral role for Semaphorin3F-Neuropilin signaling in patterning neural crest within the new vertebrate head.},
journal = {Development (Cambridge, England)},
volume = {145},
number = {14},
pages = {},
doi = {10.1242/dev.164780},
pmid = {29980564},
issn = {1477-9129},
mesh = {Animals ; *Body Patterning ; CRISPR-Cas Systems ; Cell Movement ; Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Head/*embryology ; Lampreys/genetics ; Melanocytes/cytology/metabolism ; Neural Crest/cytology/*embryology ; Neuropilins/genetics/*metabolism ; Phylogeny ; Semaphorins/genetics/*metabolism ; Sensory Receptor Cells/metabolism ; *Signal Transduction ; Skull/cytology ; },
abstract = {The origin of the vertebrate head is one of the great unresolved issues in vertebrate evolutionary developmental biology. Although many of the novelties in the vertebrate head and pharynx derive from the neural crest, it is still unknown how early vertebrates patterned the neural crest within the ancestral body plan they inherited from invertebrate chordates. Here, using a basal vertebrate, the sea lamprey, we show that homologs of Semaphorin3F (Sema3F) ligand and its Neuropilin (Nrp) receptors show complementary and dynamic patterns of expression that correlate with key periods of neural crest development (migration and patterning of cranial neural crest-derived structures). Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that lamprey Sema3F is essential for patterning of neural crest-derived melanocytes, cranial ganglia and the head skeleton, but is not required for neural crest migration or patterning of trunk neural crest derivatives. Based on comparisons with jawed vertebrates, our results suggest that the deployment of Nrp-Sema3F signaling, along with other intercellular guidance cues, was pivotal in allowing early vertebrates to organize and pattern cranial neural crest cells into many of the hallmark structures that define the vertebrate head.},
}

Animals
*Body Patterning
CRISPR-Cas Systems
Cell Movement
Gene Expression Regulation, Developmental
Gene Knockout Techniques
Head/*embryology
Lampreys/genetics
Melanocytes/cytology/metabolism
Neural Crest/cytology/*embryology
Neuropilins/genetics/*metabolism
Phylogeny
Semaphorins/genetics/*metabolism
Sensory Receptor Cells/metabolism
*Signal Transduction
Skull/cytology

@article {pmid29844184,
year = {2018},
author = {KaramiNejadRanjbar, M and Eckermann, KN and Ahmed, HMM and Sánchez C, HM and Dippel, S and Marshall, JM and Wimmer, EA},
title = {Consequences of resistance evolution in a Cas9-based sex conversion-suppression gene drive for insect pest management.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {115},
number = {24},
pages = {6189-6194},
doi = {10.1073/pnas.1713825115},
pmid = {29844184},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Ceratitis capitata/genetics ; Drosophila melanogaster/genetics ; *Evolution, Molecular ; Female ; Gene Editing ; Genes, Insect/*genetics ; Male ; Models, Genetic ; Pest Control, Biological/*methods ; Sex Determination Processes/*genetics ; },
abstract = {The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in Drosophila melanogaster to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the transformer gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in D. melanogaster and by adjusting the model for the agricultural pest Ceratitis capitata, we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy.},
}

Animals
CRISPR-Cas Systems/*genetics
Ceratitis capitata/genetics
Drosophila melanogaster/genetics
*Evolution, Molecular
Female
Gene Editing
Genes, Insect/*genetics
Male
Models, Genetic
Pest Control, Biological/*methods
Sex Determination Processes/*genetics

@article {pmid29500128,
year = {2018},
author = {Bjursell, M and Porritt, MJ and Ericson, E and Taheri-Ghahfarokhi, A and Clausen, M and Magnusson, L and Admyre, T and Nitsch, R and Mayr, L and Aasehaug, L and Seeliger, F and Maresca, M and Bohlooly-Y, M and Wiseman, J},
title = {Therapeutic Genome Editing With CRISPR/Cas9 in a Humanized Mouse Model Ameliorates α1-antitrypsin Deficiency Phenotype.},
journal = {EBioMedicine},
volume = {29},
number = {},
pages = {104-111},
doi = {10.1016/j.ebiom.2018.02.015},
pmid = {29500128},
issn = {2352-3964},
mesh = {Adenoviridae/genetics ; Animals ; *CRISPR-Cas Systems ; Cell Proliferation ; Disease Models, Animal ; *Gene Editing ; Gene Expression ; Genetic Vectors/genetics ; Humans ; Mice ; Mice, Transgenic ; *Phenotype ; Transduction, Genetic ; Transgenes ; alpha 1-Antitrypsin/blood/*genetics/metabolism ; alpha 1-Antitrypsin Deficiency/*genetics/*metabolism/pathology/therapy ; },
abstract = {α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene edited PiZ mice. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.},
}

Adenoviridae/genetics
Animals
*CRISPR-Cas Systems
Cell Proliferation
Disease Models, Animal
*Gene Editing
Gene Expression
Genetic Vectors/genetics
Humans
Mice
Mice, Transgenic
*Phenotype
Transduction, Genetic
Transgenes
alpha 1-Antitrypsin/blood/*genetics/metabolism
alpha 1-Antitrypsin Deficiency/*genetics/*metabolism/pathology/therapy

@article {pmid29207149,
year = {2018},
author = {Hou, C and Zhuang, Z and Deng, X and Xu, Y and Zhang, P and Zhu, L},
title = {Knockdown of Trio by CRISPR/Cas9 suppresses migration and invasion of cervical cancer cells.},
journal = {Oncology reports},
volume = {39},
number = {2},
pages = {795-801},
doi = {10.3892/or.2017.6117},
pmid = {29207149},
issn = {1791-2431},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques/*methods ; Guanine Nucleotide Exchange Factors/*genetics/*metabolism ; HeLa Cells ; Humans ; Lymphatic Metastasis ; Neoplasm Invasiveness ; Protein-Serine-Threonine Kinases/*genetics/*metabolism ; Signal Transduction ; Uterine Cervical Neoplasms/*genetics/metabolism ; rho-Associated Kinases/genetics/metabolism ; rhoA GTP-Binding Protein/genetics/metabolism ; },
abstract = {Triple functional domain protein (Trio) is an evolutionarily conserved protein with guanine nucleotide exchange factors that regulate different physiological processes in some types of cancer. However, the expression and function of Trio in cervical cancer are still unknown. The purpose of this study was to detect the expression of Trio in cervical cancer tissues and to evaluate its clinical value. Furthermore, the effects of the Trio on the migration and invasion of cervical cancer cells and its mechanism were investigated in vitro. The results of the present study revealed that Trio expression levels were significantly higher in most of the clinical cervical cancer samples than in adjacent tissues. The clinicopathological significance of Trio expression was also analyzed, and the results revealed that high expression levels in cervical cancer were correlated with lymph node metastasis (P=0.005). The CRISPR/Cas9 system was used to knockdown the endogenous Trio. The inhibition of Trio significantly decreased the migration and invasion abilities of cervical cancer cells. Meanwhile, levels of RhoA/ROCK signaling factors (RhoA, Rock, and p-LIMK), which contributed to cell migration and invasion, were decreased along with the inhibition of Trio. Therefore, Trio may regulate the migration and invasion of cervical cancer through the RhoA/ROCK signaling pathway.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Cell Movement
Female
Gene Expression Regulation, Neoplastic
Gene Knockdown Techniques/*methods
Guanine Nucleotide Exchange Factors/*genetics/*metabolism
HeLa Cells
Humans
Lymphatic Metastasis
Neoplasm Invasiveness
Protein-Serine-Threonine Kinases/*genetics/*metabolism
Signal Transduction
Uterine Cervical Neoplasms/*genetics/metabolism
rho-Associated Kinases/genetics/metabolism
rhoA GTP-Binding Protein/genetics/metabolism