This proposal is submitted in response to PAR-23-119: “Catalytic Tool and Technology Development in Kidney, Urologic, and Hematologic Diseases (R21 Clinical Trial Not Allowed)”. The main goal of our application is to test the feasibility and define the strategies, reagents, parameters and methodology to perform genome editing in the urothelium of live mice in order to enhance and accelerate the research of urothelial biology and diseases. Urothelium of the bladder is a frequent site of chemical and radiation damages, inflammation, infection and uncontrolled proliferation. Unlike many other epithelial tissues that are difficult to access, urothelium is readily accessible through transurethral catheterization, a standard procedure for diagnosis, monitoring and treatment of urothelium-related disorders. Despite its easy accessibility, urothelial research has been greatly hampered by the lack of efficient, cost-effective and broadly applicable technological approaches. In the past, the Principal Investigator's laboratory and those of other investigators have relied heavily on genetically engineered mouse models to investigate gene functions and the cellular and molecular mechanisms that govern urothelial growth, differentiation and disease pathogenesis. While highly useful and informative, this technological platform is inherently time-consuming, labor-intensive and costly, and consequently no longer adequate to fulfill the increasing research needs of the urothelial field. To tackle this deficiency, we have decided to test whether it is feasible to perform genome editing in vivo in urothelium of live mice by combining the easy accessibility of the bladder urothelium with the CRISPR/Cas9 technological platform. We will approach this progressively by (1) delivering gRNAs via transurethral catheterization into mice that constitutively express Cas9 to knock out, truncate or mutate a select set of genes of interest in urothelium; (2) restricting gene knockouts specifically to basal urothelial cells versus suprabasal urothelial cells using conditional Cas9 expression systems; and (3) performing gene knockouts in wild-type mice using all-in- one adenoviruses bearing Cas9 and gRNAs or using all-in-one plasmids coupled with ultrasound-guided percutaneous needle-electrode electroporation of the bladder. The success of these experiments should open doors to brand new possibilities to target genes of interest in urothelium and model a wide spectrum of urothelium-related human diseases.