Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library

Haifeng Sun, Zhaojun Wang, Limini Shen, Yeling Feng, Lu Han, Xuezhen Qian, Runde Meng, Kangming Ji, Dong Liang, Fei Zhou, Xin Lou (), Jun Zhang () and Bin Shen ()
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Haifeng Sun: Nanjing Medical University
Zhaojun Wang: Nanjing Medical University
Limini Shen: Nanjing Medical University
Yeling Feng: Nanjing Medical University
Lu Han: Nanjing Medical University
Xuezhen Qian: Nanjing Medical University
Runde Meng: Nanjing Medical University
Kangming Ji: Nanjing Medical University
Dong Liang: Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital
Fei Zhou: Suzhou Medical College of Soochow University
Xin Lou: Research Institute of Intelligent Computing, Zhejiang Lab
Jun Zhang: Nanjing Medical University
Bin Shen: Nanjing Medical University

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: Abstract DddA-derived cytosine base editors (DdCBEs) greatly facilitated the basic and therapeutic research of mitochondrial DNA mutation diseases. Here we devise a saturated spacer library and successfully identify seven DddA homologs by performing high-throughput sequencing based screen. DddAs of Streptomyces sp. BK438 and Lachnospiraceae bacterium sunii NSJ-8 display high deaminase activity with a strong GC context preference, and DddA of Ruminococcus sp. AF17-6 is highly compatible to AC context. We also find that different split sites result in wide divergence on off-target activity and context preference of DdCBEs derived from these DddA homologs. Additionally, we demonstrate the orthogonality between DddA and DddIA, and successfully minimize the nuclear off-target editing by co-expressing corresponding nuclear-localized DddIA. The current study presents a comprehensive and unbiased strategy for screening and characterizing dsDNA cytidine deaminases, and expands the toolbox for mtDNA editing, providing additional insights for optimizing dsDNA base editors.

Date: 2023
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DOI: 10.1038/s41467-023-42359-3

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