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Crispr screen

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https://www.readbyqxmd.com/read/28334828/flexible-crispr-library-construction-using-parallel-oligonucleotide-retrieval
#1
Abigail Read, Shaojian Gao, Eric Batchelor, Ji Luo
CRISPR/Cas9-based gene knockout libraries have emerged as a powerful tool for functional screens. We present here a set of pre-designed human and mouse sgRNA sequences that are optimized for both high on-target potency and low off-target effect. To maximize the chance of target gene inactivation, sgRNAs were curated to target both 5΄ constitutive exons and exons that encode conserved protein domains. We describe here a robust and cost-effective method to construct multiple small sized CRISPR library from a single oligo pool generated by array synthesis using parallel oligonucleotide retrieval...
March 16, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28334759/highly-efficient-biallelic-genome-editing-of-human-es-ips-cells-using-a-crispr-cas9-or-talen-system
#2
Kazuo Takayama, Keisuke Igai, Yasuko Hagihara, Rina Hashimoto, Morifumi Hanawa, Tetsushi Sakuma, Masashi Tachibana, Fuminori Sakurai, Takashi Yamamoto, Hiroyuki Mizuguchi
Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds...
February 21, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28333914/genome-scale-crispr-cas9-knockout-and-transcriptional-activation-screening
#3
Julia Joung, Silvana Konermann, Jonathan S Gootenberg, Omar O Abudayyeh, Randall J Platt, Mark D Brigham, Neville E Sanjana, Feng Zhang
Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28319113/combinatorial-crispr-cas9-screens-for-de-novo-mapping-of-genetic-interactions
#4
John Paul Shen, Dongxin Zhao, Roman Sasik, Jens Luebeck, Amanda Birmingham, Ana Bojorquez-Gomez, Katherine Licon, Kristin Klepper, Daniel Pekin, Alex N Beckett, Kyle Salinas Sanchez, Alex Thomas, Chih-Chung Kuo, Dan Du, Assen Roguev, Nathan E Lewis, Aaron N Chang, Jason F Kreisberg, Nevan Krogan, Lei Qi, Trey Ideker, Prashant Mali
We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies...
March 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28319085/synergistic-drug-combinations-for-cancer-identified-in-a-crispr-screen-for-pairwise-genetic-interactions
#5
Kyuho Han, Edwin E Jeng, Gaelen T Hess, David W Morgens, Amy Li, Michael C Bassik
Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs...
March 20, 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/28303677/examination-of-crispr-cas9-design-tools-and-the-effect-of-target-site-accessibility-on-cas9-activity
#6
Ciaran M Lee, Timothy H Davis, Gang Bao
The recent adaptation of the CRISPR/Cas9 system for targeted genome engineering has led to its widespread applications in many fields worldwide. In order to better understand the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design these studies have spawned a plethora of gRNA design tools with algorithms based solely on direct or indirect sequence features...
March 16, 2017: Experimental Physiology
https://www.readbyqxmd.com/read/28303292/editing-the-genome-of-hipsc-with-crispr-cas9-disease-models
#7
REVIEW
Andrew R Bassett
The advent of human-induced pluripotent stem cell (hiPSC) technology has provided a unique opportunity to establish cellular models of disease from individual patients, and to study the effects of the underlying genetic aberrations upon multiple different cell types, many of which would not normally be accessible. Combining this with recent advances in genome editing techniques such as the clustered regularly interspaced short palindromic repeat (CRISPR) system has provided an ability to repair putative causative alleles in patient lines, or introduce disease alleles into a healthy "WT" cell line...
March 16, 2017: Mammalian Genome: Official Journal of the International Mammalian Genome Society
https://www.readbyqxmd.com/read/28280930/crisprtools-a-flexible-computational-platform-for-performing-crispr-cas9-experiments-in-the-mouse
#8
Kevin A Peterson, Glen L Beane, Leslie O Goodwin, Peter M Kutny, Laura G Reinholdt, Stephen A Murray
Genome editing using the CRISPR/Cas9 RNA-guided endonuclease system has rapidly become a driving force for discovery in modern biomedical research. This simple yet elegant system has been widely used to generate both loss-of-function alleles and precision knock-in mutations using single-stranded donor oligonucleotides. Our CRISPRtools platform supports both of these applications in order to facilitate the use of CRISPR/Cas9. While there are several tools that facilitate CRISPR/Cas9 design and screen for potential off-target sites, the process is typically performed sequentially on single genes, limiting scalability for large-scale programs...
March 9, 2017: Mammalian Genome: Official Journal of the International Mammalian Genome Society
https://www.readbyqxmd.com/read/28280001/a-rapid-and-effective-method-for-screening-sequencing-and-reporter-verification-of-engineered-frameshift-mutations-in-zebrafish
#9
Sergey V Prykhozhij, Shelby L Steele, Babak Razaghi, Jason N Berman
Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing...
March 9, 2017: Disease Models & Mechanisms
https://www.readbyqxmd.com/read/28279800/crispr-cas9-system-driven-site-specific-selection-pressure-on-herpes-simplex-virus-genomes
#10
Zhihua Li, Yanwei Bi, Hongjian Xiao, Le Sun, Yuan Ren, Yadong Li, Chen Chen, Wei Cun
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...
March 6, 2017: Virus Research
https://www.readbyqxmd.com/read/28274275/whole-exome-sequencing-coupled-with-unbiased-functional-analysis-reveals-new-hirschsprung-disease-genes
#11
Hongsheng Gui, Duco Schriemer, William W Cheng, Rajendra K Chauhan, Guillermo Antiňolo, Courtney Berrios, Marta Bleda, Alice S Brooks, Rutger W W Brouwer, Alan J Burns, Stacey S Cherny, Joaquin Dopazo, Bart J L Eggen, Paola Griseri, Binta Jalloh, Thuy-Linh Le, Vincent C H Lui, Berta Luzón-Toro, Ivana Matera, Elly S W Ngan, Anna Pelet, Macarena Ruiz-Ferrer, Pak C Sham, Iain T Shepherd, Man-Ting So, Yunia Sribudiani, Clara S M Tang, Mirjam C G N van den Hout, Herma C van der Linde, Tjakko J van Ham, Wilfred F J van IJcken, Joke B G M Verheij, Jeanne Amiel, Salud Borrego, Isabella Ceccherini, Aravinda Chakravarti, Stanislas Lyonnet, Paul K H Tam, Maria-Mercè Garcia-Barceló, Robert M W Hofstra
BACKGROUND: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human...
March 8, 2017: Genome Biology
https://www.readbyqxmd.com/read/28263296/guidescan-software-for-improved-single-and-paired-crispr-guide-rna-design
#12
Alexendar R Perez, Yuri Pritykin, Joana A Vidigal, Sagar Chhangawala, Lee Zamparo, Christina S Leslie, Andrea Ventura
We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) for single- and paired-gRNA genome-wide screens. We also show that the trie data structure of GuideScan enables the design of gRNAs that are more specific than those designed by existing tools.
March 6, 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/28254804/gene-editing-with-crispr-cas9-rna-directed-nuclease
#13
REVIEW
Thomas Doetschman, Teodora Georgieva
Genetic engineering of model organisms and cultured cells has for decades provided important insights into the mechanisms underlying cardiovascular development and disease. In the past few years the development of several nuclease systems has broadened the range of model/cell systems that can be engineered. Of these, the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has become the favorite for its ease of application. Here we will review this RNA-guided nuclease system for gene editing with respect to its usefulness for cardiovascular studies and with an eye toward potential therapy...
March 3, 2017: Circulation Research
https://www.readbyqxmd.com/read/28253259/scalable-design-of-paired-crispr-guide-rnas-for-genomic-deletion
#14
Carlos Pulido-Quetglas, Estel Aparicio-Prat, Carme Arnan, Taisia Polidori, Toni Hermoso, Emilio Palumbo, Julia Ponomarenko, Roderic Guigo, Rory Johnson
CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model...
March 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28248921/development-of-improved-vaccine-cell-lines-against-rotavirus
#15
Weilin Wu, Nichole Orr-Burks, Jon Karpilow, Ralph A Tripp
Rotavirus is a major cause of severe gastroenteritis among very young children. In developing countries, rotavirus is the major cause of mortality in children under five years old, causing up to 20% of all childhood deaths in countries with high diarrheal disease burden, with more than 90% of these deaths occurring in Africa and Asia. Rotavirus vaccination mimics the first infection without causing illness, thus inducing strong and broad heterotypic immunity against prospective rotavirus infections. Two live vaccines are available, Rotarix and RotaTeq, but vaccination efforts are hampered by high production costs...
March 1, 2017: Scientific Data
https://www.readbyqxmd.com/read/28241139/transcription-control-by-the-enl-yeats-domain-in-acute-leukaemia
#16
Michael A Erb, Thomas G Scott, Bin E Li, Huafeng Xie, Joshiawa Paulk, Hyuk-Soo Seo, Amanda Souza, Justin M Roberts, Shiva Dastjerdi, Dennis L Buckley, Neville E Sanjana, Ophir Shalem, Behnam Nabet, Rhamy Zeid, Nana K Offei-Addo, Sirano Dhe-Paganon, Feng Zhang, Stuart H Orkin, Georg E Winter, James E Bradner
Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo...
March 9, 2017: Nature
https://www.readbyqxmd.com/read/28228480/a-stable-but-reversible-integrated-surrogate-reporter-for-assaying-crispr-cas9-stimulated-homology-directed-repair
#17
Yahong Wen, Grace Liao, Thomas Pritchard, Ting-Ting Zhao, Jon P Connelly, Shondra M Pruett-Miller, Valerie Blanc, Nicholas O Davidson, Blair B Madison
The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) following aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway, or alternatively, for enhancing homology directed repair (HDR) to facilitate the generation of a specific mutation (or knock-in). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA (gRNA) activity...
February 22, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28218837/rapid-and-efficient-genome-editing-in-staphylococcus-aureus-by-using-an-engineered-crispr-cas9-system
#18
Weizhong Chen, Yifei Zhang, Won-Sik Yeo, Taeok Bae, Quanjiang Ji
Staphylococcus aureus, a major human pathogen, has been the cause of serious infectious diseases with a high mortality rate. Although genetics is a key means to study S. aureus physiology, such as drug resistance and pathogenesis, genetic manipulation in S. aureus is always time-consuming and labor-intensive. Here we report a CRISPR/Cas9 system (pCasSA) for rapid and efficient genome editing, including gene deletion, insertion, and single-base substitution mutation in S. aureus. The designed pCasSA system is amenable to the assembly of spacers and repair arms by Golden Gate assembly and Gibson assembly, respectively, enabling rapid construction of the plasmids for editing...
March 2, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28215525/explicit-modeling-of-sirna-dependent-on-and-off-target-repression-improves-the-interpretation-of-screening-results
#19
Andrea Riba, Mario Emmenlauer, Amy Chen, Frederic Sigoillot, Feng Cong, Christoph Dehio, Jeremy Jenkins, Mihaela Zavolan
RNAi is broadly used to map gene regulatory networks, but the identification of genes that are responsible for the observed phenotypes is challenging, as small interfering RNAs (siRNAs) simultaneously downregulate the intended on targets and many partially complementary off targets. Additionally, the scarcity of publicly available control datasets hinders the development and comparative evaluation of computational methods for analyzing the data. Here, we introduce PheLiM (https://github.com/andreariba/PheLiM), a method that uses predictions of siRNA on- and off-target downregulation to infer gene-specific contributions to phenotypes...
February 22, 2017: Cell Systems
https://www.readbyqxmd.com/read/28211892/high-throughput-in-situ-cell-electroporation-microsystem-for-parallel-delivery-of-single-guide-rnas-into-mammalian-cells
#20
Shengtai Bian, Yicen Zhou, Yawei Hu, Jing Cheng, Xiaofang Chen, Youchun Xu, Peng Liu
Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells...
February 13, 2017: Scientific Reports
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