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

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DOI

  • Xi Xiang
  • Xiaoying Zhao
  • Xiaoguang Pan, Qingdao-Europe Advanced Institute for Life Sciences, Beijing Genomics Institute (BGI)-Qingdao, Qingdao, China.
  • ,
  • Zhanying Dong, Qingdao-Europe Advanced Institute for Life Sciences, Beijing Genomics Institute (BGI)-Qingdao, Qingdao, China.
  • ,
  • Jiaying Yu, Qingdao-Europe Advanced Institute for Life Sciences, Beijing Genomics Institute (BGI)-Qingdao, Qingdao, China., University of Chinese Academy of Sciences
  • ,
  • Siyuan Li, University of Chinese Academy of Sciences, Qingdao-Europe Advanced Institute for Life Sciences, Beijing Genomics Institute (BGI)-Qingdao, Qingdao, China.
  • ,
  • Xue Liang, Qingdao-Europe Advanced Institute for Life Sciences, Beijing Genomics Institute (BGI)-Qingdao, Qingdao, China.
  • ,
  • Peng Han, Qingdao-Europe Advanced Institute for Life Sciences, Beijing Genomics Institute (BGI)-Qingdao, Qingdao, China.
  • ,
  • Kunli Qu, Qingdao-Europe Advanced Institute for Life Sciences, Beijing Genomics Institute (BGI)-Qingdao, Qingdao, China.
  • ,
  • Jonas Brorson Jensen
  • Jean Farup
  • Fei Wang
  • Trine Skov Petersen
  • Lars Bolund
  • Huajing Teng, Peking University
  • ,
  • Lin Lin
  • Yonglun Luo
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.
OriginalsprogEngelsk
TidsskriftMolecular Therapy - Nucleic Acids
Vol/bind24
Sider (fra-til)403-415
Antal sider13
ISSN2162-2531
DOI
StatusUdgivet - jun. 2021

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