Genome-Scale CRISPR-Cas9 Knockout

Abstract

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic\rrepeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate\rgene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9\rknockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both\rnegative and positive selection screening in human cells. First, we used the GeCKO library to identify\rgenes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model,\rwe screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor.\rOur highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits\rNF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide\rRNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of\rgenome-scale screening with Cas9.