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Large-Scale Single Guide RNA Library Construction and Use for CRISPR–Cas9-Based Genetic Screens

Author(s)
Wang, Tim; Lander, Eric Steven; Sabatini, David
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Abstract
The ability to systematically disrupt genes serves as a powerful tool for understanding their function. The programmable CRISPR–Cas9 system enables efficient targeting of large numbers of genes through the use of single guide RNA (sgRNA) libraries. In cultured mammalian cells, collections of knockout mutants can be readily generated by means of transduction of Cas9–sgRNA lentiviral pools, screened for a phenotype of interest, and counted using high-throughput DNA sequencing. This technique represents the first general method for undertaking systematic loss-of-function genetic screens in mammalian cells. Here, we introduce the methodology and rationale for conducting CRISPR-based screens, focusing on distinguishing positive and negative selection strategies.
Date issued
2016-03
URI
http://hdl.handle.net/1721.1/105931
Department
Massachusetts Institute of Technology. Department of Biology; Whitehead Institute for Biomedical Research; Koch Institute for Integrative Cancer Research at MIT
Journal
Cold Spring Harbor Protocols
Publisher
Cold Spring Harbor Laboratory Press
Citation
Wang, Tim, Eric S. Lander, and David M. Sabatini. “Large-Scale Single Guide RNA Library Construction and Use for CRISPR–Cas9-Based Genetic Screens.” Cold Spring Harbor Protocols 2016.3 (2016): pdb.top086892.
Version: Author's final manuscript
ISSN
1940-3402
1559-6095

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