Genetic Screens in Human Cells Using the CRISPR-Cas9 System
Improving Whole-Genome Screens
Improved methods are needed for the knockout of individual genes in genome-scale functional screens. Wang et al. (p. 80, published online 12 December) and Shalem et al. (p. 84, published online 12 December) used the bacterial CRISPR/Cas9 system to power-screen protocols that avoid several of the pitfalls associated with small interfering RNA (siRNA) screens. Genome editing by these methods completely disrupts target genes, thus avoiding weak signals that can occur when transcript abundance is partially decreased by siRNA. Furthermore, gene targeting by the CRISPR system is more precise and appears to produce substantially fewer off-target effects than existing methods.
Abstract
The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.
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Supplementary Material
Summary
Materials and Methods
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Science
Volume 343 | Issue 6166
3 January 2014
3 January 2014
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Copyright © 2014, American Association for the Advancement of Science.
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Received: 8 October 2013
Accepted: 2 December 2013
Published in print: 3 January 2014
Acknowledgments
We thank all members of the Sabatini and Lander labs, especially J. Engreitz, S. Schwartz, A. Shishkin, and Z. Tsun for protocols, reagents, and advice; T. Mikkelsen for assistance with oligonucleotide synthesis; and L. Gaffney for assistance with figures. This work was supported by the U.S. National Institutes of Health (CA103866) (D.M.S.), National Human Genome Research Institute (2U54HG003067-10) (E.S.L.), the Broad Institute of MIT and Harvard (E.S.L.), and an award from the U.S. National Science Foundation (T.W.). The composition of the sgRNA pools and screening data can be found in the supplementary materials. A patent application has been filed by the Broad Institute relating to aspects of the work described in this manuscript. Inducible Cas9 and sgRNA backbone lentiviral vectors and the genome-scale sgRNA plasmid pool are deposited in Addgene.
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