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Cold Spring Harb Protoc. 2016 Mar 1;2016(3):pdb.top086892. doi: 10.1101/pdb.top086892.

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

Author information

1
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts 02139; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
2
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.

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.

PMID:
26933254
PMCID:
PMC4804892
DOI:
10.1101/pdb.top086892
[Indexed for MEDLINE]
Free PMC Article

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