Efficient correction of Duchenne muscular dystrophy mutations by SpCas9 and dual gRNAs

CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this ed-iting method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predic-tive repair outcome, efficient in-frame deletion of a part of DMD exon 51 was achieved in HEK293T cells with plasmids ex-pressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human DMD (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleo-proteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a hu-man MYOD transcription factor. Restoration of DMD expres-sion at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combina-tion of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.

Automated summary

CRISPR gene therapy shows promise for treating DMD by restoring dystrophin expression through efficient gene editing strategies. The successful correction of mutations and induction of myotubes in cells may provide a valuable therapeutic approach for DMD treatment.