Efficient exon skipping by base-editor-mediated abrogation of exonic splicing enhancers
Han Qiu,
Geng Li,
Juanjuan Yuan,
Dian Yang,
Yunqing Ma,
Feng Wang,
Yi Dai,
Xing Chang
Affiliations
Han Qiu
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
Geng Li
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
Juanjuan Yuan
Shunde Hospital, Southern Medical University, Foshan 528308, Guangdong, China
Dian Yang
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
Yunqing Ma
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
Feng Wang
Department of Laboratory Medicine, Ningbo Medical Center Lihuili Hospital, Ningbo 315040, Zhejiang, China
Yi Dai
Department of Neurology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100730, China
Xing Chang
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; Corresponding author
Summary: 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.
