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Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing

Author

Listed:
  • Jianhang Yin

    (Peking University)

  • Rusen Lu

    (Peking University)

  • Changchang Xin

    (Peking University)

  • Yuhong Wang

    (Peking University)

  • Xinyu Ling

    (Peking University)

  • Dong Li

    (Peking University)

  • Weiwei Zhang

    (Peking University)

  • Mengzhu Liu

    (Peking University)

  • Wutao Xie

    (Peking University)

  • Lingyun Kong

    (Peking University)

  • Wen Si

    (Peking University)

  • Ping Wei

    (Peking University)

  • Bingbing Xiao

    (Peking University First Hospital)

  • Hsiang-Ying Lee

    (Peking University)

  • Tao Liu

    (Peking University)

  • Jiazhi Hu

    (Peking University)

Abstract

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

Suggested Citation

  • Jianhang Yin & Rusen Lu & Changchang Xin & Yuhong Wang & Xinyu Ling & Dong Li & Weiwei Zhang & Mengzhu Liu & Wutao Xie & Lingyun Kong & Wen Si & Ping Wei & Bingbing Xiao & Hsiang-Ying Lee & Tao Liu & , 2022. "Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-28900-w
    DOI: 10.1038/s41467-022-28900-w
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    References listed on IDEAS

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    Cited by:

    1. Jianhang Yin & Kailun Fang & Yanxia Gao & Liqiong Ou & Shaopeng Yuan & Changchang Xin & Weiwei Wu & Wei-wei Wu & Jiaxu Hong & Hui Yang & Jiazhi Hu, 2022. "Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration," Nature Communications, Nature, vol. 13(1), pages 1-8, December.
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    3. 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.

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