Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening
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Author(s)
Joung, Julia
Konermann, Silvana M
Gootenberg, Jonathan S
Abudayyeh, Omar Osama
Platt, Randall Jeffrey
Brigham, Mark D
Sanjana, Neville E
Zhang, Feng
Date Issued
March 2017
Journal
Nature Protocols
Publisher
Springer Nature
Citation
Joung, Julia, et al. “Genome-Scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening.” Nature Protocols, vol. 12, no. 4, Mar. 2017, pp. 828–63.
Version
Author's final manuscript
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.
MIT Department
Harvard University--MIT Division of Health Sciences and Technology
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
McGovern Institute for Brain Research at MIT
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1038/NPROT.2017.016
