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Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells

Author

Listed:
  • Na Zhao

    (Guangzhou University
    Ltd.)

  • Jian Zhou

    (Guangzhou University
    The First Affiliated Hospital of Xi’an Medical University)

  • Tianfu Tao

    (Guangzhou University)

  • Qi Wang

    (Guangzhou University)

  • Jie Tang

    (Guangzhou University)

  • Dengluan Li

    (Guangzhou University)

  • Shixue Gou

    (Guangzhou National Laboratory)

  • Zhihong Guan

    (Guangzhou University)

  • Joshua Seun Olajide

    (Guangzhou University)

  • Jiejing Lin

    (Guangzhou University)

  • Shuo Wang

    (Guangzhou University)

  • Xiaoping Li

    (Sun Yat-sen University)

  • Jiankui Zhou

    (Guangzhou University)

  • Zongliang Gao

    (Guangzhou Medical University)

  • Gang Wang

    (Guangzhou University)

Abstract

Continuous directed evolution of base editors (BEs) has been successful in bacteria cells, but not yet in mammalian cells. Here, we report the development of a Continuous Directed Evolution system in Mammalian cells (CDEM). CDEM enables the BE evolution in a full-length manner with Cas9 nickase. We harness CDEM to evolve the deaminases of cytosine base editor BE3 and adenine base editors, ABEmax and ABE8e. The evolved cytidine deaminase variants on BE4 architecture show not only narrowed editing windows, but also higher editing purity and low off-target activity without a trade-off in on-targeting activity. The evolved ABEmax and ABE8e variants exhibit narrowed or shifted editing windows to different extents, and lower off-target effects. The results illustrate that CDEM is a simple but powerful approach to continuously evolve BEs without size restriction in the mammalian environment, which is advantageous over continuous directed evolution system in bacteria cells.

Suggested Citation

  • Na Zhao & Jian Zhou & Tianfu Tao & Qi Wang & Jie Tang & Dengluan Li & Shixue Gou & Zhihong Guan & Joshua Seun Olajide & Jiejing Lin & Shuo Wang & Xiaoping Li & Jiankui Zhou & Zongliang Gao & Gang Wang, 2024. "Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-52483-3
    DOI: 10.1038/s41467-024-52483-3
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    References listed on IDEAS

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    1. Jungjoon K. Lee & Euihwan Jeong & Joonsun Lee & Minhee Jung & Eunji Shin & Young-hoon Kim & Kangin Lee & Inyoung Jung & Daesik Kim & Seokjoong Kim & Jin-Soo Kim, 2018. "Directed evolution of CRISPR-Cas9 to increase its specificity," Nature Communications, Nature, vol. 9(1), pages 1-10, December.
    2. Julian Grünewald & Ronghao Zhou & Sara P. Garcia & Sowmya Iyer & Caleb A. Lareau & Martin J. Aryee & J. Keith Joung, 2019. "Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors," Nature, Nature, vol. 569(7756), pages 433-437, May.
    3. Junhao Fu & Qing Li & Xiaoyu Liu & Tianxiang Tu & Xiujuan Lv & Xidi Yin & Jineng Lv & Zongming Song & Jia Qu & Jinwei Zhang & Jinsong Li & Feng Gu, 2021. "Human cell based directed evolution of adenine base editors with improved efficiency," Nature Communications, Nature, vol. 12(1), pages 1-11, December.
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