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Science DOI:10.1126/science.1247005

Genome-scale CRISPR-Cas9 knockout screening in human cells.

Publication TypeJournal Article
Year of Publication2014
AuthorsShalem, O, Sanjana, NE, Hartenian, E, Shi, X, Scott, DA, Mikkelsen, TS, Heckl, D, Ebert, BL, Root, DE, Doench, JG, Zhang, F
JournalScience
Volume343
Issue6166
Pages84-7
Date Published2014 Jan 03
ISSN1095-9203
KeywordsAdaptor Proteins, Signal Transducing, Cell Survival, Clustered Regularly Interspaced Short Palindromic Repeats, Cullin Proteins, Drug Resistance, Neoplasm, Gene Knockout Techniques, Gene Library, Genes, Neurofibromatosis 1, Genes, Neurofibromatosis 2, Genetic Loci, Genetic Testing, Genome-Wide Association Study, Humans, Indoles, Lentivirus, Mediator Complex, Melanoma, Pluripotent Stem Cells, Protein Kinase Inhibitors, raf Kinases, Selection, Genetic, Sulfonamides, Transcription Factors
Abstract

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

URLhttp://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=24336571
DOI10.1126/science.1247005
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/24336571?dopt=Abstract

Alternate JournalScience
PubMed ID24336571
PubMed Central IDPMC4089965
Grant ListDP1 MH100706 / MH / NIMH NIH HHS / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
1DP1-MH100706 / DP / NCCDPHP CDC HHS / United States
1R01-DK097768 / DK / NIDDK NIH HHS / United States