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Science. 2014 Jan 3;343(6166):84-87. doi: 10.1126/science.1247005. Epub 2013 Dec 12.

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

Author information

1
Broad Institute of MIT and Harvard, 7 Cambridge Center, MA 02142, USA.
2
McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3
Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA.
4
Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
#
Contributed equally

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.

PMID:
24336571
PMCID:
PMC4089965
DOI:
10.1126/science.1247005
[Indexed for MEDLINE]
Free PMC Article

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