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Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016. Epub 2017 Mar 23.

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

Joung J1,2,3,4, Konermann S2,3,4, Gootenberg JS2,3,4,5, Abudayyeh OO2,3,4,6, Platt RJ2,3,4, Brigham MD2,3,4, Sanjana NE2,3,4, Zhang F1,2,3,4.

Author information

1
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
2
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
3
McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts, USA.
4
Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
5
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
6
Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Abstract

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.

PMID:
28333914
PMCID:
PMC5526071
DOI:
10.1038/nprot.2017.016
[Indexed for MEDLINE]
Free PMC Article

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