Engineering RsDddA as mitochondrial base editor with wide target compatibility and enhanced activity.

Abstract

Double-stranded DNA-specific cytidine deaminase (DddA) base editors hold great promise for applications in bio-medical research, medicine, and biotechnology. Strict sequence preference on spacing region presents a challenge for DddA editors to reach their full potential. To overcome this sequence-context constraint, we analyzed a protein dataset and identified a novel DddAtox homolog from Ruminococcus sp. AF17-6 (RsDddA). We engineered RsDddA for mitochondrial base editing in a mammalian cell line and demonstrated RsDddA-derived cytosine base editors (RsDdCBE) offered a broadened NC sequence compatibility and exhibited robust editing efficiency. Moreover, our results suggest the average frequencies of mitochondrial genome-wide off-target editing arising from RsDdCBE are comparable to canonical DdCBE and its variants.
© 2023 The Author(s).

Authors

Kai Cheng, Cao Li, Jiachuan Jin, Xuezhen Qian, Jiayin Guo, Limini Shen, Yichen Dai, Xue Zhang, Zhanwei Li, Yichun Guan, Fei Zhou, Jin Tang, Jun Zhang, Bin Shen, Xin Lou

Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China.