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

  • Nat Commun. 2025 Jul 15;16(1):6502. doi: 10.1038/s41467-025-61696-z.
Hongyu Chen  #  1 ,  Qingtong Tan  #  1  2 ,  Li Li  1 ,  Lanxin Li  3 ,  Jiqiang Fu  1 ,  Wencheng Zhu  1 ,  Jie Li  1 ,  Yining Wang  1 ,  Shiyan Li  4 ,  Huimin Li  1  2 ,  Yidi Sun  1 ,  Qiang Sun  1 ,  Zongyang Lu  5  6 ,  Zhen Liu  7  8
Affiliations
  • 1. Shanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences, Shanghai, China.
  • 2. University of Chinese Academy of Sciences, Beijing, China.
  • 3. School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
  • 4. Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
  • 5. Shanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences, Shanghai, China. [email protected].
  • 6. Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China. [email protected].
  • 7. Shanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of Sciences, Shanghai, China. [email protected].
  • 8. Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China. [email protected].
  • # Contributed equally.
PMID: 40664653 DOI: 10.1038/s41467-025-61696-z
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.

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