Project Summary A major challenge for the cancer genomics community is determining which somatic mutations are contributing to tumorigenesis and which are “passenger” (neutral) mutations. Somatic mutations that cause altered mRNA splicing have recently been appreciated as oncogenic driver events, such as the case with skipping of exon 14 in the MET proto-oncogene. Additionally, lung cancer patients with skipping of MET exon 14 respond well to targeted drug inhibitors. Through computational analysis of both DNA and mRNA sequencing data of 495 lung cancer donor samples, we have identified 635 cases of exon-skipping associated with somatic mutation;; however, we lack experimental evidence to know which of these exons might also represent oncogenic driver events. This project will test the hypothesis that a subset of exon-skipping events associated with somatic mutations are novel oncogenic alterations. To test this hypothesis, our first aim will determine if previously uncharacterized exon-skipping events, associated with somatic mutations in RAS pathway genes, are oncogenic. RAS pathway genes are known to be recurrently mutated in lung cancer and many are targetable alterations;; therefore, we will focus our initial studies on exon-skipping events which are most likely to be oncogenic and most likely to lead to a therapeutic target. Our second aim will develop a novel high- throughput CRISPR-Cas9 screen targeting all 635 exons to test if any of these exons are oncogenic when they are skipped. Our final aim will use homology-directed CRISPR-Cas9 to knock-in candidate splice mutations and validate that these mutations cause exon-skipping, which leads to an oncogenic gene alteration. Completion of this study will set the framework for more broad studies of somatic mutations that affect gene function through alternative splicing and to investigate novel targeted therapies.