Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.

Treatment of progressive and fatal genetic diseases has been one of the biggest challenges to modern medicine. Duchenne muscular dystrophy (DMD) is one such rare genetic disorder 1,2 causing severe progressive musclewasting (OMIM 310,200). It is an X chromosome linked recessive disorder that affects roughly 1/5000 male births. DMD is caused by mutations such as exon deletions, exon duplications, point mutations, insertions and splice site mutations in the dystrophin (DMD) gene 3-5 . Two therapeutic genetic approaches for DMD have reached clinical trials: reframing by exon skipping with antisense oligonucleotides (ASO) and expression of truncated DMD open reading frame (ORF) called micro-dystrophins to compensate the lack of dystrophin 6-9 . This effort has led to marketing authorization of four antisense products in the US 10 and the approval of the first micro-dystrophin product Elevidys 11 . Nevertheless, efficacy of these strategies is debated. For example, ASOs mediated exon skipping approaches have shown modest increase in dystrophin expression 12-14 , whereas the micro-dystrophin approach suffers from the fact that it is conceptually unable to compensate for a full-size dystrophin, modifying DMD to a less severe form called Becker muscular dystrophy 6 . In addition, adverse events were observed during the clinical trials such as hepatotoxicity, thrombotic microangiopathy, or cardiomyositis and death 15 . Therefore, it is imperative to explore and test other potential strategies to cure DMD.

According to the Leiden database (https:// www. dmd. nl/), exon duplications correspond to 10-15% of all mutations that induce a frameshift in the DMD ORF, resulting in premature termination. Interestingly, DMD exon duplications offer a context where it is possible to revert to a full-length dystrophin. This was initially demonstrated with the use of ASO in a mouse model with a duplication of the exon 2 15 . Subsequently, a CRISPR/Cas9 strategy employing a single guide RNA was developed, leading to the stable removal of duplications spanning exons 18-30 16 and exon 2 in immortalized myoblasts 17 , exons 55-59 in induced pluripotent stem cell (iPSC) differentiated to cardiomyocytes 18 as well as exons 18-25 19 and exons 3-16 in primary myoblasts 20 . This gene editing OPEN