Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells

被引：3773

作者：

Shalem, Ophir ^{[1
,2
]}

Sanjana, Neville E. ^{[1
,2
]}

Hartenian, Ella ^{[1
]}

Shi, Xi ^{[1
,3
]}

Scott, David A. ^{[1
,2
]}

Mikkelsen, Tarjei S. ^{[1
]}

Heckl, Dirk ^{[4
]}

Ebert, Benjamin L. ^{[4
]}

Root, David E. ^{[1
]}

Doench, John G. ^{[1
]}

Zhang, Feng ^{[1
,2
]}

机构：

[1] Broad Inst MIT & Harvard, Cambridge Ctr 7, Cambridge, MA 02142 USA

[2] MIT, Dept Biol Engn, Dept Brain & Cognit Sci, McGovern Inst Brain Res, Cambridge, MA 02139 USA

[3] Broad Inst Harvard & MIT, Stanley Ctr Psychiat Res, Cambridge Ctr 7, Cambridge, MA 02142 USA

[4] Harvard Univ, Sch Med, Brigham & Womens Hosp, Div Hematol,Dept Med, Boston, MA 02115 USA

来源：

SCIENCE | 2014年 / 343卷 / 6166期

关键词：

RNA; TRANSCRIPTION; GENE; CAS9; SPECIFICITY; SEQUENCE; BRAF;

D O I：

10.1126/science.1247005

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页码：84 / 87

页数：4

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