Correction of exon 2, exon 2–9 and exons 8–9 duplications in DMD patient myogenic cells by a single CRISPR/Cas9 system

Abstract

Abstract 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.