Abstract
The multinucleated architecture of muscle presents unique challenges for AAV-delivered CRISPR/Cas-based gene editing strategies. Because only a small subset of myonuclei are transduced by AAV, and Cas proteins traffic poorly between myonuclear domains, gene editing rates in this tissue have been extremely low. We developed a modular, compact peptide tag that when appended to Cas9 improves propagation across myonuclear domains and enhances gene editing efficiencies. Our strategy is applicable to all nuclear-targeted gene therapy cargoes and highlights the importance of considering the spatial dimension of gene regulation in the context of therapeutic strategies.
Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify “Myospreader,” a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.