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https://www.readbyqxmd.com/read/29158600/crispr-cas9-library-screening-for-drug-target-discovery
#1
REVIEW
Morito Kurata, Kouhei Yamamoto, Branden S Moriarity, Masanobu Kitagawa, David A Largaespada
CRISPR/Cas9-based tools have rapidly developed in recent years. These include CRISPR-based gene activation (CRISPRa) or inhibition (CRISPRi), for which there are libraries. CRISPR libraries for loss of function have been widely used to identify new biological mechanisms, such as drug resistance and cell survival signals. CRISPRa is highly useful in screening for gain of functions, and CRISPRi is a more powerful tool than RNA interference (RNAi) libraries in screening for loss of functions. Positive selection using a CRISPR library can detect survival cells with specific conditions, such as drug treatment, and it can easily clarify drug resistance mechanisms...
November 20, 2017: Journal of Human Genetics
https://www.readbyqxmd.com/read/29158538/pinapl-py-a-comprehensive-web-application-for-the-analysis-of-crispr-cas9-screens
#2
Philipp N Spahn, Tyler Bath, Ryan J Weiss, Jihoon Kim, Jeffrey D Esko, Nathan E Lewis, Olivier Harismendy
Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service...
November 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29156781/toxoplasma-gondii-clp-family-protein-tgclpb1-plays-a-crucial-role-in-thermotolerance
#3
Shinuo Cao, Nali Du, Heming Chen, Yu Pang, Zhaoxia Zhang, Jun Zheng, Honglin Jia
Caseinolytic peptidase B (ClpB) plays a pivotal role in suppressing and reversing protein aggregation. Toxoplasma gondii is an intracellular parasitic protozoan that infects a wide variety of mammals and birds and therefore is exposed to a broad range of living condition. We screened ToxoDB (http://ToxoDB.org) and identified 10 putative T. gondii genes encoding members of the Clp superfamily of caseinolytic proteases and chaperones. Of these, we focused on characterizing the Class I ATP-dependent molecular chaperones TgClpB1, TgClpB2, and TgClpB3...
October 17, 2017: Oncotarget
https://www.readbyqxmd.com/read/29150513/high-prevalence-and-genetic-diversity-of-large-phicd211-phicdif1296t-like-prophages-in-clostridioides-difficile
#4
Julian R Garneau, Ognjen Sekulovic, Bruno Dupuy, Olga Soutourina, Marc Monot, Louis-Charles Fortier
Clostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131-kbp. It shares morphological and genomic similarity with other large siphophages like phage 949 infecting Lactococcus lactis and phage c-st infecting Clostridium botulinum...
November 17, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/29150011/optimization-of-crispr-cas9-genome-editing-for-loss-of-function-in-the-early-chick-embryo
#5
Shashank Gandhi, Michael L Piacentino, Felipe M Vieceli, Marianne E Bronner
The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions...
December 1, 2017: Developmental Biology
https://www.readbyqxmd.com/read/29150007/crispr-in-animals-and-animal-models
#6
Ellen Shrock, Marc Güell
CRISPR-Cas9 has revolutionized the generation of transgenic animals. This system has demonstrated an unprecedented efficiency, multiplexability, and ease of use, thereby reducing the time and cost required for genome editing and enabling the production of animals with more extensive genetic modifications. It has also been shown to be applicable to a wide variety of animals, from early-branching metazoans to primates. Genome-wide screens in model organisms have been performed, accurate models of human diseases have been constructed, and potential therapies have been tested and validated in animal models...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29150006/genome-engineering-using-haploid-embryonic-stem-cells
#7
Takuro Horii, Izuho Hatada
Haploidy is a useful feature for the study of gene function because disruption of one allele in haploid cells, which contain only a single set of chromosomes, can cause loss-of-function phenotypes. Recent success in generating haploid embryonic stem (ES) cells from several mammalian species, including human, provides a new platform for simple genetic manipulation of the mammalian genome. The genome-editing potential of the CRISPR/Cas system is enhanced by the use of haploid ES cells. For example, CRISPR/Cas has been used for high-efficiency generation of multiple knockouts and knockins in haploid ES cells, with potential application in genome-wide screening...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29150005/crispr-libraries-and-screening
#8
John T Poirier
CRISPR-Cas9 technology has revolutionized large-scale functional genomic screening in mammalian cell-culture systems. Due in part to optimized lentiviral delivery vectors; it is now possible to perform CRISPR-Cas9 screens in animals in order to study biological processes in the context of a whole organism and within more physiologically relevant environment. This chapter focuses primarily on mouse models of human cancers; viral vectors used for simultaneous tumor initiation and genome editing and sgRNA library design considerations...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29150003/crispr-cas9-technology-applications-and-human-disease-modeling
#9
Marta Martinez-Lage, Raúl Torres-Ruiz, Sandra Rodriguez-Perales
The CRISPR/Cas9 system development has revolutionized the field of genome engineering through the efficient creation of targeted breaks in the DNA of almost any organism and cell type, opening an avenue for a wide range of applications in biomedical research and medicine. Apart from gene edition through knock-in or knock-out approaches, CRISPR/Cas9 technology has been used for many other purposes, including regulation of endogenous gene expression, epigenome editing, live-cell imaging of chromosomal loci, edition of RNA and high-throughput screening...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29141013/indcaps-a-tool-for-designing-screening-primers-for-crispr-cas9-mutagenesis-events
#10
Charles Hodgens, Zachary L Nimchuk, Joseph J Kieber
Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles...
2017: PloS One
https://www.readbyqxmd.com/read/29140568/crispr-cas9-based-safe-harbor-gene-editing-in-rhesus-ipscs
#11
Ravi Chandra Yada, John W Ostrominski, Ilker Tunc, So Gun Hong, Jizhong Zou, Cynthia E Dunbar
NHP iPSCs provide a unique opportunity to test safety and efficacy of iPSC-derived therapies in clinically relevant NHP models. To monitor these cells in vivo, there is a need for safe and efficient labeling methods. Gene insertion into genomic safe harbors (GSHs) supports reliable transgene expression while minimizing the risk the modification poses to the host genome or target cell. Specifically, this protocol demonstrates targeting of the adeno-associated virus site 1 (AAVS1), one of the most widely used GSH loci in the human genome, with CRISPR/Cas9, allowing targeted marker or therapeutic gene insertion in rhesus macaque induced pluripotent stem cells (RhiPSCs)...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29138316/germline-cas9-expression-yields-highly-efficient-genome-engineering-in-a-major-worldwide-disease-vector-aedes-aegypti
#12
Ming Li, Michelle Bui, Ting Yang, Christian S Bowman, Bradley J White, Omar S Akbari
The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR...
November 14, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29130157/target-discovery-for-precision-medicine-using-high-throughput-genome-engineering
#13
Xinyi Guo, Poonam Chitale, Neville E Sanjana
Over the past few years, programmable RNA-guided nucleases such as the CRISPR/Cas9 system have ushered in a new era of precision genome editing in diverse model systems and in human cells. Functional screens using large libraries of RNA guides can interrogate a large hypothesis space to pinpoint particular genes and genetic elements involved in fundamental biological processes and disease-relevant phenotypes. Here, we review recent high-throughput CRISPR screens (e.g. loss-of-function, gain-of-function, and targeting noncoding elements) and highlight their potential for uncovering novel therapeutic targets, such as those involved in cancer resistance to small molecular drugs and immunotherapies, tumor evolution, infectious disease, inborn genetic disorders, and other therapeutic challenges...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/29117169/corrigendum-genome-wide-crispr-screens-reveal-a-wnt-fzd5-signaling-circuit-as-a-druggable-vulnerability-of-rnf43-mutant-pancreatic-tumors
#14
Zachary Steinhart, Zvezdan Pavlovic, Megha Chandrashekhar, Traver Hart, Xiaowei Wang, Xiaoyu Zhang, Mélanie Robitaille, Kevin R Brown, Sridevi Jaksani, René Overmeer, Sylvia F Boj, Jarrett Adams, James Pan, Hans Clevers, Sachdev Sidhu, Jason Moffat, Stéphane Angers
This corrects the article DOI: 10.1038/nm.4219.
November 7, 2017: Nature Medicine
https://www.readbyqxmd.com/read/29109740/simple-meets-single-the-application-of-crispr-cas9-in-haploid-embryonic-stem-cells
#15
REVIEW
Zixi Yin, Lingyi Chen
The CRISPR/Cas9 system provides a powerful method for the genetic manipulation of the mammalian genome, allowing knockout of individual genes as well as the generation of genome-wide knockout cell libraries for genetic screening. However, the diploid status of most mammalian cells restricts the application of CRISPR/Cas9 in genetic screening. Mammalian haploid embryonic stem cells (haESCs) have only one set of chromosomes per cell, avoiding the issue of heterozygous recessive mutations in diploid cells. Thus, the combination of haESCs and CRISPR/Cas9 facilitates the generation of genome-wide knockout cell libraries for genetic screening...
2017: Stem Cells International
https://www.readbyqxmd.com/read/29106416/bayesian-inference-of-negative-and-positive-selection-in-human-cancers
#16
Donate Weghorn, Shamil Sunyaev
Cancer genomics efforts have identified genes and regulatory elements driving cancer development and neoplastic progression. From a microevolution standpoint, these are subject to positive selection. Although elusive in current studies, genes whose wild-type coding sequences are needed for tumor growth are also of key interest. They are expected to experience negative selection and stay intact under pressure of incessant mutation. The detection of significantly mutated (or undermutated) genes is completely confounded by the genomic heterogeneity of cancer mutation...
November 6, 2017: Nature Genetics
https://www.readbyqxmd.com/read/29091957/hepatitis-e-virus-hev-egress-role-of-bst2-tetherin-and-interferon-induced-long-non-coding-rna-lncrna-bispr
#17
Daizy Paliwal, Prashant Joshi, Subrat Kumar Panda
BACKGROUND: The biology of Hepatitis E Virus (HEV), a common cause of epidemic and sporadic hepatitis, is still being explored. HEV exits liver through bile, a process which is essential for its natural transmission by feco-oral route. Though the process of this polarised HEV egress is not known in detail, HEV pORF3 and hepatocyte actin cytoskeleton have been shown to play a role. METHODS: Our transcriptome analysis in Hepatitis E virus (HEV) replicon transfected Huh7 cells at 24 and 72 hrs indicated that at 24hrs, both LncBISPR and BST2, expressed by a bidirectional promoter were highly upregulated whereas at 72 hrs, BST2 expression was comparatively reduced accompanied by normal levels of BISPR...
2017: PloS One
https://www.readbyqxmd.com/read/29091290/combinatorial-genetics-in-liver-repopulation-and-carcinogenesis-with-a-novel-in-vivo-crispr-activation-platform
#18
Kirk J Wangensteen, Yue J Wang, Zhixun Dou, Amber W Wang, Elham Mosleh-Shirazi, Max A Horlbeck, Luke A Gilbert, Jonathan S Weissman, Shelley L Berger, Klaus H Kaestner
CRISPR/Cas9 activation (CRISPRa) systems have enabled genetic screens in cultured cells lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the dCas9(+) mouse, a Cre-inducible CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation...
November 1, 2017: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/29089570/parallel-genome-wide-screens-identify-synthetic-viable-interactions-between-the-blm-helicase-complex-and-fanconi-anemia
#19
Martin Moder, Georgia Velimezi, Michel Owusu, Abdelghani Mazouzi, Marc Wiedner, Joana Ferreira da Silva, Lydia Robinson-Garcia, Fiorella Schischlik, Rastislav Slavkovsky, Robert Kralovics, Michael Schuster, Christoph Bock, Trey Ideker, Stephen P Jackson, Jörg Menche, Joanna I Loizou
Maintenance of genome integrity via repair of DNA damage is a key biological process required to suppress diseases, including Fanconi anemia (FA). We generated loss-of-function human haploid cells for FA complementation group C (FANCC), a gene encoding a component of the FA core complex, and used genome-wide CRISPR libraries as well as insertional mutagenesis to identify synthetic viable (genetic suppressor) interactions for FA. Here we show that loss of the BLM helicase complex suppresses FANCC phenotypes and we confirm this interaction in cells deficient for FA complementation group I and D2 (FANCI and FANCD2) that function as part of the FA I-D2 complex, indicating that this interaction is not limited to the FA core complex, hence demonstrating that systematic genome-wide screening approaches can be used to reveal genetic viable interactions for DNA repair defects...
November 1, 2017: Nature Communications
https://www.readbyqxmd.com/read/29084003/a-genome-wide-comprehensive-analysis-of-alterations-in-driver-genes-in-non-small-cell-lung-cancer
#20
Jun Yi, Xiang Wei, Xinqiang Li, Lei Wan, Jiashou Dong, Rui Wang
Lung cancer is one of the most common malignancies and the leading cause of cancer-related deaths worldwide. Although many oncogenes and tumor suppressors have been uncovered in the past decades, the pathogenesis and mechanisms of lung tumorigenesis and progression are unclear. The advancement of high-throughput sequencing technique and bioinformatics methods has led to the discovery of some unknown important protein-coding genes or noncoding RNAs in human cancers. In this study, we tried to identify and validate lung cancer driver genes to facilitate the diagnosis and individualized treatment of patients with this disease...
October 27, 2017: Anti-cancer Drugs
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