Duchenne and Becker muscular dystrophies (DMD/BMD) are caused by mutations in the gene encoding dystrophin, a large protein critical for the maintenance of myofibers and cardiomyocytes. A direct approach to treating DMD would be to replace or repair the gene to enable expression of therapeutic levels of dystrophin and functional assembly of the dystrophin-glycoprotein complex. Gene replacement therapies based on systemic delivery of adeno-associated viral (AAV) vectors to delivery micro-dystrophin genes are showing encouraging promise for therapy and are being tested by several groups in pre-clinical and clinical trials. However, microdystrophins are not fully functional, and episomal AAV vectors are likely to be slowly lost during normal muscle activity and aging. This limitation could be overcome by methods to bypass or repair dystrophin mutations via gene editing. The CRISPR/Cas9 system has potential to provide highly specific gene modification, and has recently been shown to induce low levels of dystrophin in striated muscles of dystrophic mdx mice. However, one-third of all DMD cases arise by a spontaneous new mutation, such that gene editing strategies will need to be adapted for a wide spectrum of genetic lesions throughout the 2.2 megabase DMD gene. We have explored the use of AAV vectors to delivery CRISPR/Cas9 cassettes to muscles of dystrophic mdx4cv mice and show that multiple strategies can lead to expression of nearly full-length dystrophin at levels up to 25% of wild type. These preliminary results indicate that the CRISPR/Cas9 system can be adapted for in vivo use, but the approach needs considerable optimization before it could be considered therapeutically relevant. We propose to test multiple parameters of the delivery system to enhance efficiency with the goal of obtaining physiologically significant dystrophin & DGC expression without adverse events. These approaches include optimizing delivery & expression of components of the CRISPR/Cas9 system, exploring timed and limited duration delivery of Cas9, and targeting both differentiated muscle cells and their stem cell progenitors. Our initial focus will be on developing methods to achieve dystrophin gene editing at an efficiency needed to halt or reverse the pathophysiology in muscles of dystrophic mdx4cv mice. We will also adapt methods developed in mice for AAV-mediated delivery of CRISPR/Cas9 cassettes to the CXMD dog model of DMD. The ability to induce therapeutic levels of dystrophin in animal models would enable further studies to limit genotoxicity and develop approaches for clinical translation. If successful, the studies could significantly advance the potential for clinical use of gene editing for DMD and other genetic disorders of muscle.