Engineered CBEs based on Macaca fascicularis A3A with improved properties for precise genome editing
Chun-Yan Ren,
Yan-Shan Liu,
Yu-Shan He,
Lin-Pei Zhang,
Jun-Hua Rao,
Yijian Rao,
Jian-Huan Chen
Affiliations
Chun-Yan Ren
Laboratory of Genomic and Precision Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China
Yan-Shan Liu
Laboratory of Genomic and Precision Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China; Department of Pediatric Laboratory, Affiliated Children’s Hospital of Jiangnan University (Wuxi Children’s Hospital), Wuxi, Jiangsu, China
Yu-Shan He
Laboratory of Genomic and Precision Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China
Lin-Pei Zhang
Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China
Jun-Hua Rao
Joint Primate Research Center for Chronic Diseases, Jiangnan University and Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, China; Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, China; Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, China
Yijian Rao
Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China
Jian-Huan Chen
Laboratory of Genomic and Precision Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China; Joint Primate Research Center for Chronic Diseases, Jiangnan University and Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, China; Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, China; Jiangnan University-Xinshijie Eye Hospital Joint Ophthalmic Research Center, Xinshijie Eye Hospital, Wuxi, China; Corresponding author
Summary: Cytidine deaminase defines the properties of cytosine base editors (CBEs) for C-to-T conversion. Replacing the cytidine deaminase rat APOBEC1 (rA1) in CBEs with a human APOBEC3A (hA3A) improves CBE properties. However, the potential CBE application of macaque A3A orthologs remains undetermined. Our current study develops and evaluates engineered CBEs based on Macaca fascicularis A3A (mA3A). Here, we demonstrate that BE4-mA3A and its RNA-editing-derived variants exhibit improved CBE properties, except for DNA off-target activity, compared to BE3-rA1 and BE4-rA1. Unexpectedly, deleting Ser-Val-Arg (SVR) in BE4-mA3A dramatically reduces DNA and RNA off-target activities and improves editing accuracy, with on-target efficiency unaffected. In contrast, a chimeric BE4-hA3A-SVR+ shows editing efficiency increased by about 50%, with other properties unaffected. Our findings demonstrate that mA3A-based CBEs could provide prototype options with advantages over rA1- and hA3A-based CBEs for further optimization, highlighting the importance of the SVR motif in defining CBE intrinsic properties.
