| Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos |
| 论文作者 |
Chen, HY; Tan, QT; Li, L; Li, LX; Fu, JQ; Zhu, WC; Li, J; Wang, YN; Li, SY; Li, HM; Sun, YD; Sun, Q; Lu, ZY; Liu, Z |
| 期刊/会议名称 |
NATURE COMMUNICATIONS |
| 论文年度 |
2025 |
| 论文类别 |
|
| 摘要 |
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. |
| 卷 |
16 |
| 影响因子 |
15.7 |
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