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

Molecular Therapy: Nucleic Acids (Dec 2023)

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

Affiliations

Kai Cheng: 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
Cao Li: 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
Jiachuan Jin: Center for Reproductive Medicine, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
Xuezhen Qian: 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
Jiayin Guo: 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; Gusu School, Nanjing Medical University, Nanjing, Jiangsu, China
Limini Shen: 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
YiChen Dai: 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
Xue Zhang: Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China
Zhanwei Li: Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China
Yichun Guan: Center for Reproductive Medicine, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
Fei Zhou: Cambridge-Suda Genomic Resource Center, Jiangsu Key Laboratory of Neuropsychiatric Diseases Research, Medical College of Soochow University, Suzhou, China
Jin Tang: Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China; Corresponding author: Jin Tang, Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China.
Jun Zhang: 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; Corresponding author: Jun Zhang, 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.
Bin Shen: 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; Gusu School, Nanjing Medical University, Nanjing, Jiangsu, China; Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China; Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, China; Corresponding author: Bin Shen, 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.
Xin Lou: Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China; Corresponding author: Xin Lou, Research Institute of Intelligent Computing, Zhejiang Laboratory, Hangzhou, Zhejiang, China.

Journal volume & issue: Vol. 34
p. 102028

Abstract

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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.

Published in Molecular Therapy: Nucleic Acids

ISSN: 2162-2531 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Medicine: Therapeutics. Pharmacology
Website: https://www.cell.com/molecular-therapy-family/nucleic-acids/latest-content

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