Refined DNA Repair Manipulation Enables a Universal Knock-in Strategy in Mouse Embryos

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. Overall design: We constructed an RNA-Seq dataset to investigate the transcriptome-wide effects of Polq knockdown using CasRX in mouse embryos. One-cell stage embryos were microinjected at 20 hours post-hCG administration, and total RNA was extracted 10 hours later when the embryos had reached the 2-cell stage. Each sample consisted of five embryos, and four biological replicates were included for both the Polq knockdown (CasRX/KD-Polq) and control (CasRX) groups.