Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity

Jianan Li, Wenxia Yu, Shisheng Huang, Susu Wu, Liping Li, Jiankui Zhou, Yu Cao, Xingxu Huang () and Yunbo Qiao ()
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Jianan Li: ShanghaiTech University
Wenxia Yu: ShanghaiTech University
Shisheng Huang: ShanghaiTech University
Susu Wu: Guangzhou University
Liping Li: Guangzhou University
Jiankui Zhou: Guangzhou University
Yu Cao: Shanghai Jiao Tong University School of Medicine
Xingxu Huang: ShanghaiTech University
Yunbo Qiao: Guangzhou University

Nature Communications, 2021, vol. 12, issue 1, 1-8

Abstract: Abstract Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.

Date: 2021
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DOI: 10.1038/s41467-021-22519-z

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