ABSTRACT Chromosome rearrangements (structural variants) are common in cancer and they drive tumor development by altering the expression or function of oncogenes and tumor suppressor genes by copy number alterations, formation of oncogenic fusions or by alterations of DNA elements responsible for regulating cancer genes. We have developed an innovative CRISPR-based approach to specifically target structural variant junctions (SVJs) in cancer cells without harming normal cells. This approach, called KLIPP, is based on the use of a “split” enzyme approach consisting of inactivated dCas9 fused to the endonuclease Fok1 (Fok1-dCas9). To activate the Fok1 endonuclease, two Fok1-dCas9 complexes are brought together at the SVJ using SVJ- targeting guide RNAs leading to the induction of toxic DNA double strand breaks. We have obtained strong proof-of-concept for this approach in bladder cancer and other cancer cell systems both in cell cultures and in vivo. In this proposal we will perform pre-clinical efficacy testing of the Precision KLIPP Therapy approach in an orthotopic mouse model of bladder cancer. The premise for targeting bladder cancer with this approach is first, locally invasive bladder cancer is a common and deadly disease with few impactful therapeutic options. Second, bladder cancers typically show genomic instability with many hundreds of SVJs that could be targeted with our approach. Third, the bladder is ideal for local inter-vesicle delivery of the CRISPR reagents because of the ability to achieve high local concentrations and avoiding systemic effects. We will in this application pursue three specific aims: Aim #1: CRISPR screen to map potential Fok1-dCas9 binding toxicity genome-wide. Aim #2: Delivery of CRISPR reagents to bladder cells in culture. Aim #3: Assessment of efficacy of Precision KLIPP Therapy in a pre-clinical HGSC mouse model. With this R01 funding, we will be able to advance this project to the point of planning for human clinical trials.