**02.01.2017** // Megha Chandrashekhar, Donnelly Center for Cellular and Biomolecular Research, Dept. of Molecular Genetics, University of Toronto
Identifying genotype-specific cancer vulnerabilities using CRISPR-Cas9 genetic screens
The advent of CRISPR-Cas9 system provides an effective way to introduce targeted loss-of-function mutations in mammalian cells. CRISPR-Cas9 genetic screening enables the identification of cellular fitness genes that operate either globally (core fitness genes) or specifically within a particular genetic background or environmental context (context-specific fitness genes) at an unprecedented depth. In tumors, this is the foundation for the concept of synthetic lethality as genes required in tumor cells but not in adjacent normal tissues should make ideal therapeutic targets with high effectiveness and minimal side effects. Towards this goal, we have developed a second-generation CRISPR guide RNA library of 176,500 guides targeting 17,661 human protein-coding genes. We used the library to screen five human cell lines to identify genes whose knockouts induced significant fitness defects. We further characterize novel fitness genes of unknown function and find that they all likely exist in protein complexes with other essential genes. Our screens accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. We also identified a surprising and specific dependency on mitochondrial activity, which strongly supports the idea that oxidative phosphorylation dependency – a clear exception to the Warburg effect – is a targetable weakness of some tumors. Our findings demonstrate that the CRISPR-Cas9 system fundamentally alters the landscape for systematic genetics in human cells through rigorous identification of cell line essential genes, affording a high-resolution view of the genetic vulnerabilities of a cell that may represent therapeutic opportunities in cancer.
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