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https://www.readbyqxmd.com/read/28102837/precise-and-efficient-scarless-genome-editing-in-stem-cells-using-correct
#1
Dylan Kwart, Dominik Paquet, Shaun Teo, Marc Tessier-Lavigne
CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated-e.g., in pluripotent stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes-for example, in human induced pluripotent (iPS) stem cells...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28101863/lessons-from-animal-models-of-cytoplasmic-intermediate-filament-proteins
#2
Jamal-Eddine Bouameur, Thomas M Magin
Cytoplasmic intermediate filaments (IFs) represent a major cytoskeletal network contributing to cell shape, adhesion and migration as well as to tissue resilience and renewal in numerous bilaterians, including mammals. The observation that IFs are dispensable in cultured mammalian cells, but cause tissue-specific, life-threatening disorders, has pushed the need to investigate their function in vivo. In keeping with human disease, the deletion or mutation of murine IF genes resulted in highly specific pathologies...
2017: Sub-cellular Biochemistry
https://www.readbyqxmd.com/read/28098181/profiling-single-guide-rna-specificity-reveals-a-mismatch-sensitive-core-sequence
#3
Ting Zheng, Yingzi Hou, Pingjing Zhang, Zhenxi Zhang, Ying Xu, Letian Zhang, Leilei Niu, Yi Yang, Da Liang, Fan Yi, Wei Peng, Wenjian Feng, Ying Yang, Jianxin Chen, York Yuanyuan Zhu, Li-He Zhang, Quan Du
Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a "core" sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease...
January 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28097058/determining-virus-host-interactions-and-glycerol-metabolism-profiles-in-geographically-diverse-solar-salterns-with-metagenomics
#4
Abraham G Moller, Chun Liang
Solar salterns are excellent model ecosystems for studying virus-microbial interactions because of their low microbial diversity, environmental stability, and high viral density. By using the power of CRISPR spacers to link viruses to their prokaryotic hosts, we explored virus-host interactions in geographically diverse salterns. Using taxonomic profiling, we identified hosts such as archaeal Haloquadratum, Halorubrum, and Haloarcula and bacterial Salinibacter, and we found that community composition related to not only salinity but also local environmental dynamics...
2017: PeerJ
https://www.readbyqxmd.com/read/28096221/efficient-crispr-cas9-assisted-gene-targeting-enables-rapid-and-precise-genetic-manipulation-of-mammalian-neural-stem-cells
#5
Raul Bardini Bressan, Pooran Singh Dewari, Maria Kalantzaki, Ester Gangoso, Mantas Matjusaitis, Claudia Garcia-Diaz, Carla Blin, Vivien Grant, Harry Bulstrode, Sabine Gogolok, William C Skarnes, Steven M Pollard
Mammalian neural stem (NS) cell lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NS cells are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting - so critical for functional studies of embryonic stem cells - has not been exploited to date in NS cells...
January 17, 2017: Development
https://www.readbyqxmd.com/read/28092918/complete-sequence-and-organization-of-pfr260-the-bacillus-thuringiensis-inta-fr7-4-plasmid-harboring-insecticidal-genes
#6
Laura E Navas, Ariel F Amadio, Elio M Ortiz, Diego H Sauka, Graciela B Benintende, Marcelo F Berretta, Rubén O Zandomeni
We report the complete sequence and analysis of pFR260, a novel megaplasmid of 260,595 bp from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. It carries 7 insecticidal genes: 3 cry8 copies previously reported, 2 vip1, and 2 vip2. Also, it carries a gene encoding a putative atypical Cry protein. These genes are arranged in a region of approximately 105 kbp in size with characteristics of a pathogenicity island with a potential coleopteran-specific insecticide profile. DNA strand composition asymmetry, as determined by GC skew analysis, and the presence of a Rep protein involved in the initiation of replication suggest a bidirectional theta mechanism of replication...
January 17, 2017: Journal of Molecular Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28087399/advances-with-using-crispr-cas-mediated-gene-editing-to-treat-infections-with-hepatitis-b-virus-and-hepatitis-c-virus
#7
REVIEW
Buhle Moyo, Kristie Bloom, Tristan Scott, Abdullah Ely, Patrick Arbuthnot
Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. In the case of HBV persistence, the replication intermediate comprising covalently closed circular DNA (cccDNA) is particularly problematic. Licensed therapies have little effect on cccDNA and HBV replication relapses following treatment withdrawal...
January 10, 2017: Virus Research
https://www.readbyqxmd.com/read/28081148/characterization-of-multi-drug-resistant-enterococcus-faecalis-isolated-from-cephalic-recording-chambers-in-research-macaques-macaca-spp
#8
Stephanie E Woods, Mia T Lieberman, Francois Lebreton, Elise Trowel, César de la Fuente-Núñez, Joanne Dzink-Fox, Michael S Gilmore, James G Fox
Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 Enterococcus faecalis isolates. The E. faecalis isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance...
2017: PloS One
https://www.readbyqxmd.com/read/28071925/gene-editing-and-genetic-engineering-approaches-for-advanced-probiotics-a-review
#9
Ruby Yadav, Vishal Kumar, Mehak Baweja, Pratyoosh Shukla
The applications of probiotics are significant and thus resulted in need of genome analysis of probiotic strains. Various omics methods and systems biology approaches enables us to understand and optimize the metabolic processes. These techniques have increased the researcher's attention towards gut microbiome and provided a new source for the revelation of uncharacterized biosynthetic pathways which enables novel metabolic engineering approaches. In recent years, the broad and quantitative analysis of modified strains relies on systems biology tools such as in silico design which are commonly used methods for improving strain performance...
January 10, 2017: Critical Reviews in Food Science and Nutrition
https://www.readbyqxmd.com/read/28070592/therapeutic-genome-editing-with-engineered-nucleases
#10
Simone A Haas, Viviane Dettmer, Toni Cathomen
Targeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies...
January 10, 2017: Hämostaseologie
https://www.readbyqxmd.com/read/28065598/cas13b-is-a-type-vi-b-crispr-associated-rna-guided-rnase-differentially-regulated-by-accessory-proteins-csx27-and-csx28
#11
Aaron A Smargon, David B T Cox, Neena K Pyzocha, Kaijie Zheng, Ian M Slaymaker, Jonathan S Gootenberg, Omar A Abudayyeh, Patrick Essletzbichler, Sergey Shmakov, Kira S Makarova, Eugene V Koonin, Feng Zhang
CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity...
January 4, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28064188/upset-the-drosophila-homologue-of-set3-is-required-for-viability-and-the-proper-balance-of-active-and-repressive-chromatin-marks
#12
Kyle A McElroy, Youngsook Lucy Jung, Barry M Zee, Charlotte I Wang, Peter J Park, Mitzi I Kuroda
Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5). Here we show that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, we generated S2 cells which are mutant for upset We found that loss of UpSET is tolerated in S2 cells, but that heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone post-translational modification quantification...
January 6, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28062037/single-molecule-insight-into-target-recognition-by-crispr-cas-complexes
#13
M Rutkauskas, A Krivoy, M D Szczelkun, C Rouillon, R Seidel
Ribonucleoprotein (RNP) complexes from CRISPR-Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base pairing. For CRISPR-Cas systems that target double-stranded DNA this results in local DNA unwinding and formation of a so-called R-loop structure. Here we provide an overview how this target recognition mechanism can be dissected in great detail at the level of a single molecule...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28057934/the-driving-force-of-prophages-and-crispr-cas-system-in-the-evolution-of-cronobacter-sakazakii
#14
Haiyan Zeng, Jumei Zhang, Chensi Li, Tengfei Xie, Na Ling, Qingping Wu, Yingwang Ye
Cronobacter sakazakii is an important foodborne pathogens causing rare but life-threatening diseases in neonates and infants. CRISPR-Cas system is a new prokaryotic defense system that provides adaptive immunity against phages, latter play an vital role on the evolution and pathogenicity of host bacteria. In this study, we found that genome sizes of C. sakazakii strains had a significant positive correlation with total genome sizes of prophages. Prophages contributed to 16.57% of the genetic diversity (pan genome) of C...
January 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28052722/road-to-the-future-of-systems-biotechnology-crispr-cas-mediated-metabolic-engineering-for-recombinant-protein-production
#15
Amir Roointan, Mohammad Hossein Morowvat
The rising potential for CRISPR-Cas-mediated genome editing has revolutionized our strategies in basic and practical bioengineering research. It provides a predictable and precise method for genome modification in a robust and reproducible fashion. Emergence of systems biotechnology and synthetic biology approaches coupled with CRISPR-Cas technology could change the future of cell factories to possess some new features which have not been found naturally. We have discussed the possibility and versatile potentials of CRISPR-Cas technology for metabolic engineering of a recombinant host for heterologous protein production...
January 4, 2017: Biotechnology & Genetic Engineering Reviews
https://www.readbyqxmd.com/read/28045163/crispr-cas9-technology-applications-in-genome-engineering-development-of-sequence-specific-antimicrobials-and-future-prospects
#16
REVIEW
César de la Fuente-Núñez, Timothy K Lu
The development of CRISPR-Cas9 technology has revolutionized our ability to edit DNA and to modulate expression levels of genes of interest, thus providing powerful tools to accelerate the precise engineering of a wide range of organisms. In addition, the CRISPR-Cas system can be harnessed to design "precision" antimicrobials that target bacterial pathogens in a DNA sequence-specific manner. This capability will enable killing of drug-resistant microbes by selectively targeting genes involved in antibiotic resistance, biofilm formation and virulence...
January 3, 2017: Integrative Biology: Quantitative Biosciences From Nano to Macro
https://www.readbyqxmd.com/read/28041849/inhibition-of-crispr-cas9-with-bacteriophage-proteins
#17
Benjamin J Rauch, Melanie R Silvis, Judd F Hultquist, Christopher S Waters, Michael J McGregor, Nevan J Krogan, Joseph Bondy-Denomy
Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages...
January 12, 2017: Cell
https://www.readbyqxmd.com/read/28034840/establishment-of-porcine-xist-knockout-model-using-crispr-cas9-system
#18
Li Guoling, Zhong Cuili, Ni Sheng, Liu Dewu, Cai Gengyuan, Li Zicong, Yang Huaqiang, Wu Zhenfang
Somatic cell nuclear transfer technique has great applications in livestock breeding, production of genetically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this technique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that it may directly or indirectly affects cloning efficiency...
December 20, 2016: Yi Chuan, Hereditas
https://www.readbyqxmd.com/read/28027431/from-classical-mutagenesis-to-nuclease-based-breeding-directing-natural-dna-repair-for-a-natural-end-product
#19
Michael Pacher, Holger Puchta
The production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damages in an error-prone way. In case of radiation, multiple double strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site directed nucleases (SDNs), also referred to as sequence specific nucleases (SSNs), like the CRISPR/Cas system, enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at predefined sites in plant genomes...
December 27, 2016: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/28017588/mutations-in-cas9-enhance-the-rate-of-acquisition-of-viral-spacer-sequences-during-the-crispr-cas-immune-response
#20
Robert Heler, Addison V Wright, Marija Vucelja, David Bikard, Jennifer A Doudna, Luciano A Marraffini
CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection...
January 5, 2017: Molecular Cell
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