2. Academic Validation
  3. Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos

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

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. zylu@ion.ac.cn.
  • 6 Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China. zylu@ion.ac.cn.
  • 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. zliu2010@ion.ac.cn.
  • 8 Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China. zliu2010@ion.ac.cn.
  • # 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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