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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 sgRNAs varies greatly, although similar editing efficiency. 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 re-orients NHEJ repair toward 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE297792 | GEO | 2025/05/26

REPOSITORIES: GEO

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