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Abstract
Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.
Improved cytosine base editors are created by repurposing an adenine deaminase.
Details
; Zhu, Biyun 1 ; Ru, Gaomeng 1 ; Meng, Haowei 2
; Yan, Yongchang 2 ; Hong, Mengjia 1 ; Zhang, Dan 1 ; Luan, Changming 1 ; Zhang, Shun 1 ; Wu, Hao 2 ; Gao, Hongyi 1 ; Bai, Sijia 1 ; Li, Changqing 1 ; Ding, Ruoyi 1 ; Xue, Niannian 1 ; Lei, Zhixin 2 ; Chen, Yuting 3 ; Guan, Yuting 1
; Siwko, Stefan 4 ; Cheng, Yiyun 1 ; Song, Gaojie 1
; Wang, Liren 1 ; Yi, Chengqi 2
; Liu, Mingyao 5
; Li, Dali 1
1 East China Normal University, Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, Shanghai, China (GRID:grid.22069.3f) (ISNI:0000 0004 0369 6365)
2 Peking University, School of Life Sciences, Beijing, China (GRID:grid.11135.37) (ISNI:0000 0001 2256 9319)
3 CAS Key Laboratory of Quantitative Engineering Biology, Center for Genome Engineering and Therapy, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China (GRID:grid.458489.c) (ISNI:0000 0001 0483 7922)
4 Texas A&M University, Institute of Biosciences and Technology, Houston, USA (GRID:grid.264756.4) (ISNI:0000 0004 4687 2082)
5 East China Normal University, Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, Shanghai, China (GRID:grid.22069.3f) (ISNI:0000 0004 0369 6365); BRL Medicine, Inc., Shanghai, China (GRID:grid.22069.3f)