A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility

Junfan Guo; Wenxia Yu; Min Li; Hongyu Chen; Jie Liu; Xiaowen Xue; Jianxiang Lin; Shisheng Huang; Wenjie Shu; Xingxu Huang; Zhen Liu; Shengqi Wang; Yunbo Qiao

doi:10.1016/j.molcel.2023.04.012

A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility

Mol Cell. 2023 May 18;83(10):1710-1724.e7. doi: 10.1016/j.molcel.2023.04.012.

Authors

Junfan Guo¹, Wenxia Yu², Min Li³, Hongyu Chen⁴, Jie Liu⁵, Xiaowen Xue⁶, Jianxiang Lin⁷, Shisheng Huang⁸, Wenjie Shu⁹, Xingxu Huang¹⁰, Zhen Liu¹¹, Shengqi Wang¹², Yunbo Qiao¹³

Affiliations

¹ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
² School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China; WLA Laboratories, Shanghai 201208, China.
³ Shanghai Institute of Precision Medicine, Shanghai 200125, China; Precise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, China.
⁴ Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China.
⁵ Guangzhou Medical University, Guangzhou 511436, China.
⁶ Precise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou 510006, China.
⁷ Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China; Shanghai Institute of Precision Medicine, Shanghai 200125, China.
⁸ Zhejiang Lab, Hangzhou 311121, Zhejiang, China.
⁹ Bioinformatics Center of AMMS, Beijing 100850, China.
¹⁰ Zhejiang Lab, Hangzhou 311121, Zhejiang, China; Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou 310029, China.
¹¹ Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China. Electronic address: zliu2010@ion.ac.cn.
¹² Bioinformatics Center of AMMS, Beijing 100850, China. Electronic address: sqwang@bmi.ac.cn.
¹³ Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China; Shanghai Institute of Precision Medicine, Shanghai 200125, China. Electronic address: ybqiao@shsmu.edu.cn.

PMID: 37141888
DOI: 10.1016/j.molcel.2023.04.012

Abstract

Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddA_tox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddA_tox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddA_tox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddA_tox- or riDddA_tox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddA_tox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.

Keywords: DdCBE; Roseburia intestinalis; mitoCBE; mitochondrial DNA; mtDNA; non-TC; nuclear DNA; transactivator.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems
Cytosine
DNA, Mitochondrial / genetics
Gene Editing*
Mice
Mutation
Trans-Activators* / metabolism

Substances

Trans-Activators
Cytosine
DNA, Mitochondrial