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

View Article: PubMed Central - PubMed

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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3042-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Validation of the screen by knock-out of TBK1 and TRIB2. aLeft 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
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Fig5: Validation of the screen by knock-out of TBK1 and TRIB2. aLeft 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

Mentions: Finally, we wanted to describe unexpected dependencies in these cell lines. The tank-binding kinase TBK1 was among the strongest hits identified by the two different analysis approaches in the HCC-827 cell line (Fig. 5a, b). TBK1 was at position 5 in the sgRNA-based analysis and at position 1 in the gene-based analysis (Fig. 5a). TBK1 was described as co-synthetic lethal in KRAS mutant lung cancer [19] but, so far, it has not been associated with EGFR mutant cancer. We found that sgRNAs targeting TBK1 decrease cell viability for HCC-827 (Fig. 5b). Efficacy for knock-out of different sgRNA correlated with the degree of decrease of cell viability (Fig. 5b). In addition, in a colony formation assay, knock-down of TBK1 by different sgRNAs significantly reduced ability of cells to form colonies (Fig. 5c). TBK1 shows a strong expression in the HCC-827 cell line similarly to EGFR (Additional file 6: Table S5). We observed that many potential hits are strongly expressed (Additional file 6: Table S5). Two recent studies observed that CRISPR/Cas9 screens may generate false-positive hits for genes with high copy numbers or genes in amplified regions [20, 21]. The authors describe that DNA breaks by CRISPR/Cas9 in amplified regions cause an antiproliferative effect independent of the respective gene targeted by the sgRNA [20]. Using the canSAR database [22], we indeed found that TBK1 has 10 copy number variants in the parental HCC-827 cell line. While further studies are needed to prove that TBK1 is a true dependency, we can conclude that these experimental data validate the findings of our CRISPR screen.Fig. 5


Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening
Validation of the screen by knock-out of TBK1 and TRIB2. aLeft 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
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Fig5: Validation of the screen by knock-out of TBK1 and TRIB2. aLeft 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
Mentions: Finally, we wanted to describe unexpected dependencies in these cell lines. The tank-binding kinase TBK1 was among the strongest hits identified by the two different analysis approaches in the HCC-827 cell line (Fig. 5a, b). TBK1 was at position 5 in the sgRNA-based analysis and at position 1 in the gene-based analysis (Fig. 5a). TBK1 was described as co-synthetic lethal in KRAS mutant lung cancer [19] but, so far, it has not been associated with EGFR mutant cancer. We found that sgRNAs targeting TBK1 decrease cell viability for HCC-827 (Fig. 5b). Efficacy for knock-out of different sgRNA correlated with the degree of decrease of cell viability (Fig. 5b). In addition, in a colony formation assay, knock-down of TBK1 by different sgRNAs significantly reduced ability of cells to form colonies (Fig. 5c). TBK1 shows a strong expression in the HCC-827 cell line similarly to EGFR (Additional file 6: Table S5). We observed that many potential hits are strongly expressed (Additional file 6: Table S5). Two recent studies observed that CRISPR/Cas9 screens may generate false-positive hits for genes with high copy numbers or genes in amplified regions [20, 21]. The authors describe that DNA breaks by CRISPR/Cas9 in amplified regions cause an antiproliferative effect independent of the respective gene targeted by the sgRNA [20]. Using the canSAR database [22], we indeed found that TBK1 has 10 copy number variants in the parental HCC-827 cell line. While further studies are needed to prove that TBK1 is a true dependency, we can conclude that these experimental data validate the findings of our CRISPR screen.Fig. 5

View Article: PubMed Central - PubMed

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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3042-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus