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

Author

Listed:
  • 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)

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.

Suggested Citation

  • Haifeng Sun & Zhaojun Wang & Limini Shen & Yeling Feng & Lu Han & Xuezhen Qian & Runde Meng & Kangming Ji & Dong Liang & Fei Zhou & Xin Lou & Jun Zhang & Bin Shen, 2023. "Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-42359-3
    DOI: 10.1038/s41467-023-42359-3
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    References listed on IDEAS

    as
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