Large-Scale Single Guide RNA Library Construction and Use for CRISPR–Cas9-Based Genetic Screens
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Lander_Large-scale.pdf
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657.77 KB
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Author(s)
Wang, Tim
Lander, Eric Steven
Sabatini, David
Date Issued
March 2016
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
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Whitehead Institute for Biomedical Research
Koch Institute for Integrative Cancer Research at MIT
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1101/pdb.top086892
