Correction of the Exon 2, Exon 2-8 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 guideRNA 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. RNA sequencing of the corrected differentiated myogenic derivatives indicated rescue of dystrophin related molecular pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we demonstrated a single guide CRISPR strategy that can be utilized to delete multi-exons duplication in patient DMD genes without significant off target effect, leading to restoration of transcriptome in the corrected patient derived cells. This study further expands the application of single guide CRISPR strategy as a potential therapeutic approach for patients with larger DNA region duplication in DMD patients. To remove the exonic DMD duplication , we perfomed a transfection of single guide RNA system with CRISPR in 3 different cell lines (myoblasts containing 3 different types of DMDduplications) We then performe comparative gene expression profilling between the correted myotubes and the duplicated ones after differenciation of myoblasts into myotubes for 8 days.