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Cytosine base editing systems with minimized off-target effect and molecular size

Author

Listed:
  • Ang Li

    (Kobe University)

  • Hitoshi Mitsunobu

    (Kobe University
    Bio Palette inc)

  • Shin Yoshioka

    (Kobe University)

  • Takahisa Suzuki

    (Kobe University
    Tokyo Metropolitan University)

  • Akihiko Kondo

    (Kobe University
    Kobe University)

  • Keiji Nishida

    (Kobe University
    Kobe University)

Abstract

Cytosine base editing enables the installation of specific point mutations without double-strand breaks in DNA and is advantageous for various applications such as gene therapy, but further reduction of off-target risk and development of efficient delivery methods are desired. Here we show structure-based rational engineering of the cytosine base editing system Target-AID to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 is eliminated and additional mutations are introduced to restore enzyme function. The resulting tCDA1EQ is effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized RNA-mediated editing and gRNA-dependent/independent DNA off-targets, as assessed in human cells. Combining with the smaller Cas9 ortholog system (SaCas9), a cytosine base editing system is created that is within the size limit of AAV vector.

Suggested Citation

  • Ang Li & Hitoshi Mitsunobu & Shin Yoshioka & Takahisa Suzuki & Akihiko Kondo & Keiji Nishida, 2022. "Cytosine base editing systems with minimized off-target effect and molecular size," Nature Communications, Nature, vol. 13(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-32157-8
    DOI: 10.1038/s41467-022-32157-8
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    References listed on IDEAS

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    1. Alexis C. Komor & Yongjoo B. Kim & Michael S. Packer & John A. Zuris & David R. Liu, 2016. "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature, Nature, vol. 533(7603), pages 420-424, May.
    2. Erica McGrath & Hyunsu Shin & Linyi Zhang & Je-Nie Phue & Wells W. Wu & Rong-Fong Shen & Yoon-Young Jang & Javier Revollo & Zhaohui Ye, 2019. "Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
    3. Yi Yu & Thomas C. Leete & David A. Born & Lauren Young & Luis A. Barrera & Seung-Joo Lee & Holly A. Rees & Giuseppe Ciaramella & Nicole M. Gaudelli, 2020. "Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
    4. F. Ann Ran & Le Cong & Winston X. Yan & David A. Scott & Jonathan S. Gootenberg & Andrea J. Kriz & Bernd Zetsche & Ophir Shalem & Xuebing Wu & Kira S. Makarova & Eugene V. Koonin & Phillip A. Sharp & , 2015. "In vivo genome editing using Staphylococcus aureus Cas9," Nature, Nature, vol. 520(7546), pages 186-191, April.
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    Cited by:

    1. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    2. Jian Wang & Ke Wang & Zhe Deng & Zhiyu Zhong & Guo Sun & Qing Mei & Fuling Zhou & Zixin Deng & Yuhui Sun, 2024. "Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces," Nature Communications, Nature, vol. 15(1), pages 1-16, December.

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