Genome-scale CRISPR-Cas9 knockout screening in human cells

Ophir Shalem; Neville E Sanjana; Ella Hartenian; Xi Shi; David A Scott; Tarjei Mikkelson; Dirk Heckl; Benjamin L Ebert; David E Root; John G Doench; Feng Zhang

doi:10.1126/science.1247005

Genome-scale CRISPR-Cas9 knockout screening in human cells

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

Authors

Ophir Shalem^#^{1

2}, Neville E Sanjana^#^{1

2}, Ella Hartenian¹, Xi Shi^{1

3}, David A Scott^{1

2}, Tarjei Mikkelson¹, Dirk Heckl⁴, Benjamin L Ebert⁴, David E Root¹, John G Doench¹, Feng Zhang^{1

2}

Affiliations

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Signal Transducing / 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
Melanoma / genetics*
Pluripotent Stem Cells / metabolism*
Protein Kinase Inhibitors / therapeutic use
Selection, Genetic
Sulfonamides / therapeutic use
Transcription Factors / genetics
Vemurafenib
raf Kinases / antagonists & inhibitors

Substances

Adaptor Proteins, Signal Transducing
CUL3 protein, human
Cullin Proteins
Indoles
MED12 protein, human
Mediator Complex
Protein Kinase Inhibitors
Sulfonamides
TADA2B protein, human
Transcription Factors
Vemurafenib
raf Kinases

Abstract

Publication types

MeSH terms

Substances

Grants and funding