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Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells (Science 3 January 2014)
2014-10-19 15:30 written by ercsb (2023-12-11 21:21 Modify)
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Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells


The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2CUL3TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.


Editor's summary

Improving Whole-Genome Screens

Improved methods are needed for the knockout of individual genes in genome-scale functional screens.Wang et al. (p. 80, published online 12 December) and Shalem et al. (p. 84, published online 12 December) used the bacterial CRISPR/Cas9 system to power-screen protocols that avoid several of the pitfalls associated with small interfering RNA (siRNA) screens. Genome editing by these methods completely disrupts target genes, thus avoiding weak signals that can occur when transcript abundance is partially decreased by siRNA. Furthermore, gene targeting by the CRISPR system is more precise and appears to produce substantially fewer off-target effects than existing methods.





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