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Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos

Author

Listed:
  • Hongyu Chen

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences)

  • Qingtong Tan

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Li Li

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences)

  • Lanxin Li

    (ShanghaiTech University)

  • Jiqiang Fu

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences)

  • Wencheng Zhu

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences)

  • Jie Li

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences)

  • Yining Wang

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences)

  • Shiyan Li

    (University of Chinese Academy of Sciences, Chinese Academy of Sciences)

  • Huimin Li

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Yidi Sun

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences)

  • Qiang Sun

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences)

  • Zongyang Lu

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences
    Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology)

  • Zhen Liu

    (Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences
    Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology)

Abstract

The design and screening of sgRNA in CRISPR-dependent gene knock-in is always laborious. Therefore, a universal and highly efficient knock-in strategy suitable for different sgRNA target sites is necessary. In our mouse embryo study, we find that the knock-in efficiency guided by adjacent sgRNAs varies greatly, although similar indel frequency. MMEJ-biased sgRNAs usually lead to high knock-in efficiency, whereas NHEJ-biased sgRNAs result in low knock-in efficiency. Blocking MMEJ repair by knocking down Polq can enhance knock-in efficiency, but inhibiting NHEJ repair shows variable effects. We identify a compound, AZD7648, that can shift DSBs repair towards MMEJ. Finally, by combining AZD7648 treatment with Polq knockdown, we develop a universal and highly efficient knock-in strategy in mouse embryos. This approach is validated at more than ten genomic loci, achieving up to 90% knock-in efficiency, marking a significant advancement toward predictable and highly efficient CRISPR-mediated gene integration.

Suggested Citation

  • Hongyu Chen & Qingtong Tan & Li Li & Lanxin Li & Jiqiang Fu & Wencheng Zhu & Jie Li & Yining Wang & Shiyan Li & Huimin Li & Yidi Sun & Qiang Sun & Zongyang Lu & Zhen Liu, 2025. "Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos," Nature Communications, Nature, vol. 16(1), pages 1-15, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-61696-z
    DOI: 10.1038/s41467-025-61696-z
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    References listed on IDEAS

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    1. Grzegorz Ira & Achille Pellicioli & Alitukiriza Balijja & Xuan Wang & Simona Fiorani & Walter Carotenuto & Giordano Liberi & Debra Bressan & Lihong Wan & Nancy M. Hollingsworth & James E. Haber & Marc, 2004. "DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1," Nature, Nature, vol. 431(7011), pages 1011-1017, October.
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