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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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.
DepartmentDavid H. Koch Institute for Integrative Cancer Research at MIT; Massachusetts Institute of Technology. Department of Biology; Whitehead Institute for Biomedical Research
Cold Spring Harbor Protocols
Cold Spring Harbor Laboratory Press
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.
Author's final manuscript