Journal Article

Engineering a precise adenine base editor with minimal bystander editing

Liu, Meizhen, Zhang, Shun, Chen, Liang, Zhang, Xiaohui, Wu, Hao, Ru, Gaomeng, Bai, Sijia, Meng, Haowei, Liu, Mingyao, Luan, Changming, Yi, Chengqi, Li, Dali, Zhang, Dan, Gao, Hongyi, Hong, Mengjia, Cheng, Yiyun, Huang, Yifan, Xue, Niannian, Yang, Jing, Zhu, Biyun, Zhao, Yongxiang, Wang, Liren, Song, Gaojie, Zhong, Liping

2023

Overview

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1–2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A 5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA–target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals. A precise adenine base editor variant, ABE9, was developed to generate single adenine transition at pathogenic homopolymeric adenine sites with minimal DNA/RNA off-target effects, suggesting promising potential for gene therapeutics.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/92/469 / 631/92/610 / 631/92/612 / Adenine / Adenosine / Animals / Biochemical Engineering / Biochemistry / Bioorganic Chemistry / Cell Biology / Chemistry / Chemistry and Materials Science / Chemistry/Food Science / CRISPR-Cas Systems - genetics / Cytosine / Deoxyribonucleic acid / DNA / Editing / Embryos / Gene Editing / Localization / Mice / Mutation / Nuclear safety / Nucleotide sequence / Nucleotides / Rats / Ribonucleic acid / RNA / RNA, Guide, CRISPR-Cas Systems / Safety engineering