Genome-wide CRISPR Screening in Cancer Cells

Cancer cell lines were transduced with a lentiviral vector expressing the Cas9 nuclease under blasticidin selection (pXPR-311Cas9). Each Cas9-expressing cell line was subjected to a Cas9 activity assay^{3 (link)} to characterize the efficacy of CRISPR/Cas9 in these cell lines (Supplementary Table 1). Cell lines with less than 45% measured Cas9 activity were considered ineligible for screening. Stable polyclonal Cas9+ cell lines were then infected in replicate (n = 3) at low multiplicity of infection (MOI < 1) with a library of 76,106 unique sgRNAs (Avana), which after filtering out sex chromosomes was composed of 70,086 targeting 17,670 genes (~4 sgRNAs per gene) annotated in the consensus coding sequence (CCDS) database, and 995 non-targeting control sgRNAs (Supplementary Table 2). Cells were selected in puromycin and blasticidin for 7 days and then passaged without selection while maintaining a representation of 500 cells per sgRNA until 21 days after infection. Genomic DNA was purified from endpoint cell pellets, the sgRNA barcodes are PCR amplified with sufficient gDNA to maintain representation, and the PCR products are sequenced using standard Illumina machines and protocols.