Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos
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 s...
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61696-z |
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| author | 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 |
| author_facet | 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 |
| author_sort | Hongyu Chen |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-2c86be5d05b24cf9bcfb4aaa3c12c1ae |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-2c86be5d05b24cf9bcfb4aaa3c12c1ae2025-08-20T03:05:14ZengNature PortfolioNature Communications2041-17232025-07-0116111510.1038/s41467-025-61696-zRefined DNA repair manipulation enables a universal knock-in strategy in mouse embryosHongyu Chen0Qingtong Tan1Li Li2Lanxin Li3Jiqiang Fu4Wencheng Zhu5Jie Li6Yining Wang7Shiyan Li8Huimin Li9Yidi Sun10Qiang Sun11Zongyang Lu12Zhen Liu13Shanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesSchool of Life Science and Technology, ShanghaiTech UniversityShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesShanghai Key Laboratory of Precision Gene Editing and Clinical Translation, Institute of Neuroscience, Center for Excellence in Brain Science & Intelligence Technology, Chinese Academy of SciencesAbstract 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.https://doi.org/10.1038/s41467-025-61696-z |
| spellingShingle | 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 Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos Nature Communications |
| title | Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos |
| title_full | Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos |
| title_fullStr | Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos |
| title_full_unstemmed | Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos |
| title_short | Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos |
| title_sort | refined dna repair manipulation enables a universal knock in strategy in mouse embryos |
| url | https://doi.org/10.1038/s41467-025-61696-z |
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