Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening

Abstract

Background: Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge.

Results: To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation.

Conclusions: We show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.

Keywords: Driver mutations; Dropout; EGFR; Kinase; NRAS; Negative selection; Whole genome CRISPR screen.

Fig. 1

Representation of whole genome sgRNA library at different time points. a Schematic representation of the negative loss-of-function screen using lung cancer cell line HCC-827 and neuroblastoma cell line CHP-212. b Cumulative frequency of sgRNAs by deep sequencing at control time point (day −10), day 14, day 21, and day 28 for HCC-827 cell line. Shift in the curves at days 14, 21, and 28 represents the depletion of essential sgRNAs. Each time point was measured in duplicates. c Same as in b) but for CHP-212 cell line. d Plots of normalized sgRNA reads for HCC-827 cell line at time points day 14, day 21, and day 28. Dark colored dots represent the 1 000 non-targeting control sgRNAs and light colored dots represent the 57 096 targeting sgRNAs. Each time point was measured in duplicates and log2 of median fold changes versus the control time point (day −10) are represented. e Same as in d) but for CHP-212 cell line. f Gene ontology terms describing sgRNAs and genes whose knockdown causes under-representation of HCC-827 cells at day 14. g Gene ontology terms describing sgRNAs and genes whose knockdown cause under-representation of CHP-212 cells at day 14

Fig. 2

sgRNAs depleted in the whole genome screen. a Scatterplot representing fold changes of the 57 096 targeting sgRNAs in the HCC-827 cell line at day 14 and day 21. Fold changes at day 14 or day 21 were calculated compared to the control time point at day −10. All time points were measured in duplicates and median fold changes are shown. Dark green colored dots represent the 1 000 non-targeting control sgRNAs and grey colored dots represent the 57 096 targeting sgRNAs. Genes of interest were annotated by the software Spotfire and visualization was further enhanced by red colored dots. b Same as in a) but for CHP-212 cell line. c Scatterplot of fold changes of 1 571 kinases in the HCC-827 cell line at time points day 14 and day 21 versus control time point day −10. d Same as c) but for HCC-827 cells. e, f Scatterplots for Q1 and RSA down of 57 096 targeting sgRNAs in the HCC-827 (e) and CHP-212 cell lines (f) at time point day 14. Dark green colored dots represent the 1 000 non-targeting control sgRNAs. g, h Scatterplot for Q1 and RSA down of the 1 571 sgRNAs for kinases are shown for the HCC-827 (e) and CHP-212 (f) cell lines

Fig. 3

Depletion of kinases EGFR and MAP2K1 correlates with sensitivity towards EGFR and MEK inhibitors. a left panel: HCC-827 and CHP-212 cells were treated with indicated concentrations of Gefitinib for 72 h. Then, cell viability was measured by Cell Titer Glo according to the manufacturer’s instructions. Middle panel: Fold change for the three independent sgRNAs for EGFR from the screen are depicted at time point day 14. Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of Gefitinib. Then, cells were lysed and analysed by Western blot. b same as a) but the MEK inhibitor AZD6244 was used instead (left panel) and fold change for sgRNAs for MAP2K1 and MAP2K3 from the screen are depicted (middle panel). Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of AZD6244. Then, cells were lysed and analysed by Western blot

Fig. 4

Validation of target kinases by inhibitors. a left panel: HCC-827 and CHP-212 cells were treated with indicated concentrations of CHEK1 inhibitor AZD7762 for 72 h. Then, cell viability was measured by Cell Titer Glo according to the manufacturer’s instructions. Middle panel: Fold changes for three independent sgRNAs for CHEK1 inhibitor AZD7762 are depicted at time point day 14. b Same as a) but CDK1,2,5,7,9 inhibitor and respective sgRNAs are shown. c Same as a) but AKT1/2/inhibitor MK2206 and respective sgRNAs are shown. d Same as a) but FGFR inhibitor BGJ398 and respective sgRNAs are shown. e Same as a) but BRAF inhibitor Vemurafenib and respective sgRNAs are shown

Fig. 5

Validation of the screen by knock-out of TBK1 and TRIB2. a Left side: Scatterplot of fold changes for the 1 462 kinase sgRNAs of the HCC-827 cell line at time point day 14 and time point day 21 versus control time point day −10. sgRNAs against TBK1 were annotated by the software Spotfire and visualization was further enhanced by red colored dots. Rigth side: Scatterplot of Q1 and RSA down for the 1 462 kinase sgRNAs of the HCC-827 cell line at time point day 14 versus control time point day −10. b HCC-827 cell line was transduced with a non-targeting sgRNA against GFP and 5 different sgRNAs against TBK1 (TBK1_2 is a sgRNA included in the genome-wide screens while the other 4 were newly designed for this validation). Cell viability was measured 18days after viral transduction. c Equal number of cells transduced with a non-targeting sgRNA against GFP and 5 different sgRNAs against TBK1. Crystal violet staining was performed after 28 days. d-e Same as in a-c but CHP212 cells were used with the candidate TRIB2