Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder most commonly caused by out-of-frame mutations in the dystrophin gene that result in the absence of functional dystrophin protein and lead to progressive skeletal and cardiac muscle failure. A therapeutically attractive strategy is to excise the mutational hotspot spanning exons 45–55 to restore the reading frame and generate an internally deleted yet functional dystrophin, a genotype–phenotype relationship supported by milder Becker muscular dystrophy presentations. Achieving efficient, precise large genomic deletions in post-mitotic muscle, however, remains a key technical challenge for in vivo genome editing.
Here we describe a sticky-end CRISPR genome editing platform engineered to improve large-fragment excision by generating sticky DNA ends that favor productive re-ligation following dual-cut deletion. Our proprietary sticky-end CRISPR system is designed for in vivo delivery and can be packaged within a single adeno-associated virus (AAV), enabling streamlined administration. In patient-derived induced pluripotent stem cell (iPSC) differentiated myogenic cells, sticky-end CRISPR produced high-efficiency deletion of the DMD exon 45–55 region, yielding robust reframing at the transcript level and restoration of dystrophin protein expression. To evaluate in vivo feasibility, we delivered editor components using either AAV or lipid nanoparticles (LNP) into a humanized DMD mouse model and observed generation of the intended exon 45–55 deletion across relevant muscle tissues. Together, these results establish the feasibility of sticky-end CRISPR–enabled large deletions for DMD reframing and support further development of this AAV- and LNP-deliverable approach as a broadly applicable therapeutic strategy for patients with mutations in the 45–55 exon region.