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

Xi Xiang, Xiaoying Zhao, Xiaoguang Pan, Zhanying Dong, Jiaying Yu, Siyuan Li, Xue Liang, Peng Han, Kunli Qu, Jonas Brorson Jensen, Jean Farup, Fei Wang, Trine Skov Petersen, Lars Bolund, Huajing Teng, Lin Lin, Yonglun Luo

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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 editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of DMD exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human DMD (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human MYOD transcription factor. Restoration of DMD expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.

Original languageEnglish
JournalMolecular Therapy - Nucleic Acids
Pages (from-to)403-415
Number of pages13
Publication statusPublished - 4 Jun 2021


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