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

Michael K Kiessling,Sebastian Bergling,Klaus Seuwen,Walter Carbone,Tewis Bouwmeester,Guglielmo Roma,Sven Schuierer,Judith Knehr,Gerhard Rogler,Joelle Tchinda,Cheryl De Vallière,Martin Beibel,Silke Stertz

doi:10.1186/s12864-016-3042-2

Michael K Kiessling, Sebastian Bergling + Show 11 more

Open Access

https://doi.org/10.1186/s12864-016-3042-2

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Abstract

BackgroundGenome-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.ResultsTo 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.ConclusionsWe show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3042-2) contains supplementary material, which is available to authorized users.

Highlights

Genome-wide Clustered regularly interspaced short palindrome repeats (CRISPR)-Cas9 dropout screens can identify genes whose knockout affects cell viability
CRISPR-Cas9 screen and identification of essential genes involved in fundamental cellular processes To investigate whether pooled whole-genome CRISPRCas9 screening is an appropriate means to identify oncogenic drivers and novel dependencies we selected two human cancer cell lines with known mutations: (1) the neuroblastoma-derived cell line CHP-212, which carries a RAS (NRAS) Q61K mutation and is highly sensitive to MEK inhibitors [12, 13]; (2) the lung cancer cell line HCC-827, which carries a deletion in the epidermal growth factor receptor (EGFR) delE746 and is sensitive to EGFR inhibitors including Gefitinib and Erlotinib
We found that the overall technical variability across the screen was low as the distribution of non-targeting versus targeting single guide RNA (sgRNA) was consistent across all time points within each cell line (Fig. 1d, e)

Summary

Introduction

Genome-wide CRISPR-Cas dropout screens can identify genes whose knockout affects cell viability. Genome-wide CRISPR-Cas screens in a setting of positive selection have discovered gene mutations that confer drug resistance, resistance to bacterial toxins and genes involved in metastasis [1, 4, 5, 7, 10]. Using this approach Chen et al discovered key genes involved in early, late and metastatic cancer [1].

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