Publication Date:
2013-12-18
Description:
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.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089965/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089965/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Shalem, Ophir -- Sanjana, Neville E -- Hartenian, Ella -- Shi, Xi -- Scott, David A -- Mikkelsen, Tarjei S -- Heckl, Dirk -- Ebert, Benjamin L -- Root, David E -- Doench, John G -- Zhang, Feng -- 1DP1-MH100706/DP/NCCDPHP CDC HHS/ -- 1R01-DK097768/DK/NIDDK NIH HHS/ -- DP1 MH100706/MH/NIMH NIH HHS/ -- R01 DK097768/DK/NIDDK NIH HHS/ -- New York, N.Y. -- Science. 2014 Jan 3;343(6166):84-7. doi: 10.1126/science.1247005. Epub 2013 Dec 12.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24336571" target="_blank"〉PubMed〈/a〉
Keywords:
Adaptor Proteins, Signal Transducing/genetics
;
Caspase 9/*genetics
;
Cell Survival/*genetics
;
*Clustered Regularly Interspaced Short Palindromic Repeats
;
Cullin Proteins/genetics
;
Drug Resistance, Neoplasm/*genetics
;
Gene Knockout Techniques
;
Gene Library
;
Genes, Neurofibromatosis 1
;
Genes, Neurofibromatosis 2
;
Genetic Loci
;
Genetic Testing/*methods
;
Genome-Wide Association Study
;
Humans
;
Indoles/therapeutic use
;
Lentivirus
;
Mediator Complex/genetics
;
Melanoma/drug therapy/*genetics
;
Pluripotent Stem Cells/*metabolism
;
Protein Kinase Inhibitors/therapeutic use
;
Selection, Genetic
;
Sulfonamides/therapeutic use
;
Transcription Factors/genetics
;
raf Kinases/antagonists & inhibitors
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
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