Engineered IscB-ωRNA system with improved base editing efficiency for disease correction via single AAV delivery in mice
Ruochen Guo,
Xiaozhi Sun,
Feizuo Wang,
Dingyi Han,
Qiaoxia Yang,
Hua Gao,
Zhifang Li,
Zhuang Shao,
Jinqi Shi,
Rongrong Yang,
Xiaona Huo,
Junda Yan,
Guoling Li,
Qingquan Xiao,
Yuanhua Liu,
Senfeng Zhang,
Xinyu Liu,
Yingsi Zhou,
Leyun Wang,
Chunyi Hu,
Chunlong Xu
Affiliations
Ruochen Guo
Lingang Laboratory, Shanghai, China; Institute of Neuroscience, Key Laboratory of Primate Neurobiology, Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Xiaozhi Sun
Lingang Laboratory, Shanghai, China; School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China
Feizuo Wang
Department of Biological Sciences, Department of Biochemistry, Precision Medicine Translational Research Programme (TRP), National University of Singapore, Singapore, Singapore
Dingyi Han
Institute of Neuroscience, Key Laboratory of Primate Neurobiology, Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Qiaoxia Yang
Xiamen Key Laboratory of Cardiovascular Diseases, Xiamen Cardiovascular Hospital, Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, School of Medicine, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, China
Hua Gao
Lingang Laboratory, Shanghai, China; School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China
Zhifang Li
Lingang Laboratory, Shanghai, China
Zhuang Shao
Lingang Laboratory, Shanghai, China
Jinqi Shi
Lingang Laboratory, Shanghai, China
Rongrong Yang
Lingang Laboratory, Shanghai, China; Shanghai Center for Brain Science and Brain-Inspired Technology, Shanghai, China
Xiaona Huo
Lingang Laboratory, Shanghai, China; Shanghai Center for Brain Science and Brain-Inspired Technology, Shanghai, China
Junda Yan
Lingang Laboratory, Shanghai, China
Guoling Li
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Qingquan Xiao
Institute of Neuroscience, Key Laboratory of Primate Neurobiology, Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Yuanhua Liu
Institute of Neuroscience, Key Laboratory of Primate Neurobiology, Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Senfeng Zhang
Department of Biological Sciences, Department of Biochemistry, Precision Medicine Translational Research Programme (TRP), National University of Singapore, Singapore, Singapore
Xinyu Liu
Institute of Neuroscience, Key Laboratory of Primate Neurobiology, Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Yingsi Zhou
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Leyun Wang
Xiamen Key Laboratory of Cardiovascular Diseases, Xiamen Cardiovascular Hospital, Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, State Key Laboratory of Cellular Stress Biology, School of Medicine, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian, China; Corresponding author
Chunyi Hu
Department of Biological Sciences, Department of Biochemistry, Precision Medicine Translational Research Programme (TRP), National University of Singapore, Singapore, Singapore; Corresponding author
Chunlong Xu
Lingang Laboratory, Shanghai, China; School of Life Sciences and Technology, ShanghaiTech University, Shanghai, China; Shanghai Center for Brain Science and Brain-Inspired Technology, Shanghai, China; Corresponding author
Summary: IscBs, as hypercompact ancestry proteins of Cas9 nuclease, are suitable for in vivo gene editing via single adeno-associated virus (AAV) delivery. Due to the low activity of natural IscBs in eukaryotic cells, recent studies have been focusing on improving OgeuIscB’s gene editing efficiency via protein engineering. However, in vivo gene editing efficacy of IscBs for disease correction remained to be demonstrated. Here, we showed effective gene knockout and base editing in mouse embryos. To further improve IscB activity, we performed systematic engineering of IscB-associated ωRNA and identified a variant, ωRNA∗-v2, with enhanced gene editing efficiency. Furthermore, our study demonstrated the efficacy of an engineered IscB-ωRNA system for robust gene knockout and base editing in vivo. Single AAV delivery of IscB-derived cytosine and adenine base editors achieved disease correction in a mouse model of tyrosinemia. Therefore, our results indicated the great potential of miniature IscBs for developing single-AAV-based gene editing therapeutics.
