Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing

作者：Chen, Liang; Zhu, Biyun; Ru, Gaomeng; Meng, Haowei; Yan, Yongchang; Hong, Mengjia; Zhang, Dan; Luan, Changming; Zhang, Shun; Wu, Hao; Gao, Hongyi; Bai, Sijia; Li, Changqing; Ding, Ruoyi; Xue, Niannian; Lei, Zhixin; Chen, Yuting; Guan, Yuting; Siwko, Stefan; Cheng, Yiyun; Song, Gaojie; Wang, Liren; Yi, Chengqi^*; Liu, Mingyao^*; Li, Dali^*

来源：Nature Biotechnology, 2023, 41(5): 663-+.

DOI：10.1038/s41587-022-01532-7

• OFF-TARGET
• GENOMIC DNA
• MUTATIONS

Cytosine base editors (CBEs) efficiently generate precise C center dot G-to-T center dot 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 center dot G-to-G center dot 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.

• 单位
  北京大学; 中国科学院