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; Zhen Liu

doi:10.1038/s41467-025-61696-z

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.

Authors

Hongyu Chen^#¹, Qingtong Tan^#^{1

2}, Li Li¹, Lanxin Li³, Jiqiang Fu¹, Wencheng Zhu¹, Jie Li¹, Yining Wang¹, Shiyan Li⁴, Huimin Li^{1

2}, Yidi Sun¹, Qiang Sun¹, 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. 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.

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.

MeSH terms

Animals
CRISPR-Cas Systems / genetics
DNA Breaks, Double-Stranded
DNA End-Joining Repair / genetics
DNA Repair* / genetics
DNA-Directed DNA Polymerase / genetics
DNA-Directed DNA Polymerase / metabolism
Embryo, Mammalian* / metabolism
Female
Gene Editing / methods
Gene Knock-In Techniques* / methods
Mice
RNA, Guide, CRISPR-Cas Systems / genetics

Substances

RNA, Guide, CRISPR-Cas Systems
DNA-Directed DNA Polymerase