Correction of Exon 2, Exon 2-9 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

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 to 9, and exons 8 to 9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the corrected myotubes indicated rescue of dystrophin related molecular 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. This resulted in the restoration of dystrophin expression and reversal of transcriptome pathological signatures in the corrected patient-derived cells. Our study contributes valuable insights into the safety and efficacy about the application of the single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.