Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells
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Zhang_Genome-Scale.pdf
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919.95 KB
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Author(s)
Hartenian, E.
Shi, X.
Mikkelsen, T. S.
Heckl, D.
Ebert, B. L.
Root, D. E.
Doench, J. G.
Shalem, Ophir
Sanjana, Neville E
Scott, David Arthur
Date Issued
January 2014
Journal
Science
Publisher
American Association for the Advancement of Science (AAAS)
Citation
Shalem, O. "Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells." Science 343, 6166 (January 2014): 84-87 © 2014 American Association for the Advancement of Science (AAAS)
Version
Author's final manuscript
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.
MIT Department
McGovern Institute for Brain Research at MIT
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1126/science.1247005
