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

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

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-61696-z

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