Correction of three prominent mutations in mouse and human models of Duchenne muscular dystrophy by single-cut genome editing

Abstract Duchenne muscular dystrophy (DMD), one of the most common neuromuscular disorders of children, is caused by the absence of dystrophin protein in striated muscle. Deletions of exons 43, 45 and 52 represent mutational “hot spot” regions in the dystrophin gene. We created three new DMD mouse models harboring deletions of exons 43, 45 or 52 to represent common DMD mutations. To optimize CRISPR/Cas9 genome editing using the single-cut strategy, we identified single guide RNAs (sgRNAs) capable of restoring dystrophin expression by inducing exon skipping and reframing. Intramuscular delivery of AAV9 encoding SpCas9 and selected sgRNAs efficiently restored dystrophin expression in these new mouse models, offering a platform for future studies of dystrophin gene correction therapies. To validate the therapeutic potential of this approach, we identified sgRNAs capable of restoring dystrophin expression by the single-cut strategy in cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) with each of these hot spot deletion mutations. We found that the potential effectiveness of individual sgRNAs in correction of DMD mutations cannot be predicted a priori, highlighting the importance of sgRNA design and testing as a prelude for applying gene editing as a therapeutic strategy for DMD.