Gene editing is moving towards the clinic, but several challenges must be addressed in order for it to be applied to skeletal muscle disease. Adeno-associated virus (AAV) is one of the most promising technologies to enable delivery of gene editing machinery to skeletal muscle, but at least four major hurdles stand in the way of using it to safely and effectively edit genes in this tissue. These include 1) maximizing delivery of editing enzymes to muscle, 2) maximizing spread of editing enzymes across nuclei of the myofiber syncytium, 3) avoiding sustained expression of editing enzymes, and 4) integrating solutions to each of these problems in a single AAV so as to minimize total viral dose required. Given that exciting new myotropic capsids that have emerged in the literature, this proposal is primarily focused on addressing the latter 3 issues. We have previously developed ways to shuttle Cas enzymes throughout myofibers and now propose to use creative new approaches that rely on tried- and-true mechanisms to access and edit even more myonuclei in skeletal muscle. We have also developed ways to use small molecules to control AAV-delivered gene expression and propose to further develop this technology to achieve fully “off” transgene states with maximally “on” states when desired, so that Cas enzyme expression can be turned off when no longer needed. Finally, we will integrate these technologies with the latest miniaturized Cas enzymes and package all components, including guide RNAs, into a single AAV vector. We will test approaches in mouse models of myotonic dystrophy and Duchenne muscular dystrophy. Ultimately, the insights we make here will be apply to gene editing for any skeletal muscle disease and enable the next generation of therapies for muscular dystrophies.