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

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

doi:10.1016/j.omtn.2023.09.005

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

Mol Ther Nucleic Acids. 2023 Sep 9:34:102028. doi: 10.1016/j.omtn.2023.09.005. eCollection 2023 Dec 12.

Authors

Kai Cheng¹, Cao Li¹, Jiachuan Jin², Xuezhen Qian¹, Jiayin Guo^{1

3}, Limini Shen¹, YiChen Dai¹, Xue Zhang⁴, Zhanwei Li⁴, Yichun Guan², Fei Zhou⁵, Jin Tang⁴, Jun Zhang¹, Bin Shen^{1

3

4

6}, Xin Lou⁴

Affiliations

¹ State Key Laboratory of Reproductive Medicine, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing Medical University, Nanjing, Jiangsu, China.
² Center for Reproductive Medicine, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
³ Gusu School, Nanjing Medical University, Nanjing, Jiangsu, China.
⁴ Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China.
⁵ Cambridge-Suda Genomic Resource Center, Jiangsu Key Laboratory of Neuropsychiatric Diseases Research, Medical College of Soochow University, Suzhou, China.
⁶ Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, China.

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 DddA_tox 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.

Keywords: DdCBE; MT: RNA/DNA Editing; base editing; mitochondrial disease; mtDNA engineering; target compatibility.