RRC ID 62288
著者 Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F.
タイトル Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.
ジャーナル Nat Protoc
Abstract 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. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.
巻・号 12(4)
ページ 828-863
公開日 2017-4-1
DOI 10.1038/nprot.2017.016
PII nprot.2017.016
PMID 28333914
PMC PMC5526071
MeSH Animals CRISPR-Cas Systems / genetics* Gene Knockout Techniques / methods* Gene Library Genetic Vectors / genetics Genomics / methods* Humans Lentivirus / genetics Mice Phenotype Transcriptional Activation*
IF 10.419
リソース情報
ヒト・動物細胞 HUDEP-2(RCB4557)