RESUMO
Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.
Assuntos
Sistemas CRISPR-Cas , Desoxirribonuclease I/metabolismo , Endonucleases/metabolismo , Genoma Bacteriano , Animais , Sequência de Bases , Desoxirribonuclease I/genética , Embrião de Mamíferos , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Camundongos , Mutação , RNA BacterianoRESUMO
UNLABELLED: The rapid development of CRISPR-Cas9 mediated genome editing techniques has given rise to a number of online and stand-alone tools to find and score CRISPR sites for whole genomes. Here we describe the Wellcome Trust Sanger Institute Genome Editing database (WGE), which uses novel methods to compute, visualize and select optimal CRISPR sites in a genome browser environment. The WGE database currently stores single and paired CRISPR sites and pre-calculated off-target information for CRISPRs located in the mouse and human exomes. Scoring and display of off-target sites is simple, and intuitive, and filters can be applied to identify high-quality CRISPR sites rapidly. WGE also provides a tool for the design and display of gene targeting vectors in the same genome browser, along with gene models, protein translation and variation tracks. WGE is open, extensible and can be set up to compute and present CRISPR sites for any genome. AVAILABILITY AND IMPLEMENTATION: The WGE database is freely available at www.sanger.ac.uk/htgt/wge CONTACT: : vvi@sanger.ac.uk or skarnes@sanger.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Bases de Dados Factuais , Regulação da Expressão Gênica , Vetores Genéticos , Genoma , Edição de RNA/genética , Animais , Humanos , Camundongos , SoftwareAssuntos
Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Animais , Cruzamento , Proteínas Associadas a CRISPR/genética , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.