Developing ABEmax-NG with Precise Targeting and Expanded Editing Scope to Model Pathogenic Splice Site Mutations In Vivo

Shisheng Huang; Zhaodi Liao; Xiangyang Li; Zhen Liu; Guanglei Li; Jianan Li; Zongyang Lu; Yu Zhang; Xiajun Li; Xu Ma; Qiang Sun; Xingxu Huang

doi:10.1016/j.isci.2019.05.008

Developing ABEmax-NG with Precise Targeting and Expanded Editing Scope to Model Pathogenic Splice Site Mutations In Vivo

iScience. 2019 May 31:15:640-648. doi: 10.1016/j.isci.2019.05.008. Epub 2019 May 11.

Authors

Shisheng Huang¹, Zhaodi Liao², Xiangyang Li¹, Zhen Liu³, Guanglei Li⁴, Jianan Li¹, Zongyang Lu¹, Yu Zhang⁵, Xiajun Li⁵, Xu Ma⁶, Qiang Sun⁷, Xingxu Huang⁸

Affiliations

¹ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; University of Chinese Academy of Sciences, Beijing 100049, China.
² Institute of Neuroscience, Chinese Academy of Sciences (CAS) Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
³ Institute of Neuroscience, Chinese Academy of Sciences (CAS) Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China.
⁴ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; National Research Institute for Family Planning, No. 12 Dahuishi Road, Beijing 100081, China.
⁵ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
⁶ National Research Institute for Family Planning, No. 12 Dahuishi Road, Beijing 100081, China. Electronic address: nfpcc_ma@163.com.
⁷ Institute of Neuroscience, Chinese Academy of Sciences (CAS) Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China. Electronic address: qsun@ion.ac.cn.
⁸ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai 200031, China. Electronic address: huangxx@shanghaitech.edu.cn.

Abstract

RNA splicing is related to many human diseases; however, lack of efficient genetic approaches to modulate splicing has prevented us from dissecting their functions in human diseases. Recently developed base editors (BEs) offer a new strategy to modulate RNA splicing by converting conservative splice sites, but it is limited by the editing precision and scope. To overcome the limitations of currently available BE-based tools, we combined SpCas9-NG with ABEmax to generate a new BE, ABEmax-NG. We demonstrated that ABEmax-NG performed precise A⋅T to G⋅C conversion with an expanded scope, thus covering many more splicing sites. Taking advantage of this tool, we precisely achieved A⋅T to G⋅C conversion exactly at the splice sites. We further modeled pathogenic RNA splicing in vitro and in vivo. Taken together, we successfully generated a versatile tool suitable for precise and broad editing at the splice sites.

Keywords: Biological Sciences; Biological Sciences Tools; Molecular Biology.