Genome-scale CRISPR-Cas9 knockout screening in human cells

Abstract

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 NF2, CUL3, TADA2B, 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.

Fig. 1. Lentiviral delivery of Cas9 and sgRNA provides efficient depletion of target genes

(A) Lentiviral expression vector for Cas9 and sgRNA (lentiCRISPR). Puromycin selection rev marker (puro), psi packaging signal (psi+), response element (RRE), central polypurine tract (cPPT), elongation factor-1α short promoter (EFS), 2A self-cleaving peptide (P2A), and posttranscriptional regulatory element (WPRE). (B) Distribution of fluorescence from 293T-EGFP cells transduced by EGFP-targeting lentiCRISPR (sgRNAs 1-6, outlined peaks) and Cas9-only (green-shaded peak) vectors, and non-fluorescent 293T cells (gray shaded peak). (C) Distribution of fluorescence from 293T-EGFP cells transduced by EGFP-targeting shRNA (shRNAs 1-4, outlined peaks) and control shRNA (green-shaded peak) vectors, and non-fluorescent 293T cells (gray shaded peak).

Fig. 2. GeCKO library design and application for genome-scale negative selection screening

(A) Design ofsgRNA library for genome-scale knockout of coding sequences in human cells (supplementary discussion). (B and C) Cumulative frequency of sgRNAs 3 and 14 days post transduction in A375 and hES cells respectively. Shift in the 14 day curve represents the depletion in a subset of sgRNAs. (D and E) Five most significantly depleted gene sets in A375 cells p < 10⁻⁵, FDR-corrected q < 10⁻⁵) and HUES62 cells (nominal p < 10⁻⁵, FDR-corrected q < 10⁻³) indentified by Gene Set Enrichment Analysis (DSEA) (15).

Fig. 3. GeCKO screen in A375 melanoma cells reveals genes whose loss confers vemurafenib (PLX) resistance

(A ) Timeline of PLX resistance screen in A375 melanoma cells. (B) Growth of A375 cells when treated with DMSO or PLX over 14 days. (C) Boxplot showing the distribution of sgRNA frequencies at different time points, with and without PLX treatment (vehicle = DMSO). The box extends from the first to the third quartile with the whiskers denoting 1.5 times the interquartile range. Enrichment of specific sgRNAs: 7 days of PLX treatment, 1 sgRNA greater than 10-fold enrichment; 14 days of PLX treatment, 379 and 49 sgRNAs greater than 10-fold and 100-fold enrichment respectively. (D) Rank correlation of normalized sgRNA read count between biological replicates and treatment conditions. (E) Scatterplot showing enrichment of specific sgRNAs after PLX treatment. (F) Identification of top candidate genes using the RNAi Gene Enrichment Ranking (RIGER) P value analysis.

Fig. 4. Comparison of GeCKO and shRNA screens and validation of neurofibromin 2 (NF2)

(A RIGER p values for the top 100 hits from GeCKO and shRNA (19) screens for genes whose loss results in PLX resistance. Analysis using the Redudant siRNA Activity (RSA) algorithm shows a similar trend (fig. S9). (B) For the top 10 RIGER hits, the percent of unique sgRNAs (top) or shRNAs (bottom) targeting each gene that are in top 5% of all enriched sgRNAs or shRNAs. (C) Deep sequencing analysis of lentiCRISPR-mediated indel at the NF2 locus. (D) A375 cells transduced with NF2-targeting lentiCRISPR and shRNA vectors both show a decrease in NF2 protein levels. (E) Dose response curves for A375 cells transduced with individual NF2-targeting lentiCRISPR or shRNA vectors. Controls were EGFP-targeting lentiCRISPR or null hairpin shRNA vectors. Cells transduced with NF2-targeting lentiCRISPRs show a significant increase (F_1,8 = 30.3, p < 0.001, n = 4 replicates) in the half maximal effective concentration (EC₅₀) whereas cells transduced with NF2-targeting shRNA vectors do not (F_1,8 = 0.47, p = 0.51, n = 4 replicates).