TY - JOUR
T1 - Efficient correction of Duchenne muscular dystrophy mutations by SpCas9 and dual gRNAs
AU - Xiang, Xi
AU - Zhao, Xiaoying
AU - Pan, Xiaoguang
AU - Dong, Zhanying
AU - Yu, Jiaying
AU - Li, Siyuan
AU - Liang, Xue
AU - Han, Peng
AU - Qu, Kunli
AU - Jensen, Jonas Brorson
AU - Farup, Jean
AU - Wang, Fei
AU - Petersen, Trine Skov
AU - Bolund, Lars
AU - Teng, Huajing
AU - Lin, Lin
AU - Luo, Yonglun
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
U2 - 10.1016/j.omtn.2021.03.005
DO - 10.1016/j.omtn.2021.03.005
M3 - Journal article
C2 - 33868784
VL - 24
SP - 403
EP - 415
JO - Molecular Therapy - Nucleic Acids
JF - Molecular Therapy - Nucleic Acids
SN - 2162-2531
ER -