Efficient exon skipping by base-editor-mediated abrogation of exonic splicing enhancers

  • Cell Rep. 2023 Oct 30;42(11):113340. doi: 10.1016/j.celrep.2023.113340.
Han Qiu  1 ,  Geng Li  2 ,  Juanjuan Yuan  3 ,  Dian Yang  2 ,  Yunqing Ma  2 ,  Feng Wang  4 ,  Yi Dai  5 ,  Xing Chang  6
Affiliations
  • 1. Research Center for Industries of the Future, Westlake University, Hangzhou 310024, Zhejiang, China; Center for Genome Editing, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, Zhejiang, China; Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou 310024, Zhejiang, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, Zhejiang, China; Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
  • 2. Research Center for Industries of the Future, Westlake University, Hangzhou 310024, Zhejiang, China; Center for Genome Editing, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, Zhejiang, China; Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou 310024, Zhejiang, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, Zhejiang, China.
  • 3. Shunde Hospital, Southern Medical University, Foshan 528308, Guangdong, China.
  • 4. Department of Laboratory Medicine, Ningbo Medical Center Lihuili Hospital, Ningbo 315040, Zhejiang, China.
  • 5. Department of Neurology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100730, China.
  • 6. Research Center for Industries of the Future, Westlake University, Hangzhou 310024, Zhejiang, China; Center for Genome Editing, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, Zhejiang, China; Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou 310024, Zhejiang, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, Zhejiang, China. Electronic address: [email protected].
PMID: 37906593 DOI: 10.1016/j.celrep.2023.113340
Abstract

Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the Dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated Dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients' induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and Other RNA mis-splicing diseases.

Keywords
CP: Genomics; CRISPR; DMD; base editor; exon skipping; exonic splicing enhancer; gene therapy; genome editing; muscular dystrophy.
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