Fig. 2: The ssDNA donor-mediated targeted method also exhibits higher knock-in efficiency with MMEJ-biased sgRNA.

A Schematic of three sgRNAs targeting the mouse Sod1-A4 and Sod1-G93 loci. “Stop” marks the stop codon. “d” indicates the distance (in bp) from the sgRNA cut site (blue triangle) to the donor DNA insertion site (green triangle), as shown. B Indel frequencies at sgRNA target sites within the Sod1-A4 and Sod1-G93 loci. Each dot represents a single blastocyst from three independent experiments. Statistical significance was determined using a two-sided Kruskal–Wallis test. C The pie chart illustrates the distribution of DNA repair pattern (N/M = NHEJ: MMEJ). D A schematic representation of CRISPR/Cas9-mediated ssDNA gene knock-in. E Percentage of gene knock-in embryos out of the total embryos using different sgRNAs. F HDR repair proportions per knock-in embryo. Fractions above each group show HDR-positive over total injected embryos (n/N). G Distribution of DNA repair outcomes (HDR, random indels, WT) across all blastocysts analyzed in (F), summarized per sgRNA to show overall repair profiles. H A schematic representation of the classification criteria for different sgRNAs. I The “N/M” ratio of each indicated sgRNA. J Percentage of gene knock-in embryos out of the total embryos across sgRNAs categorized as MMEJ-Bias or NHEJ-Bias. K HDR repair proportion per gene knock-in embryo at indicated loci for different sgRNAs. L Correlation between knock-in efficiency and indel/MMEJ/NHEJ repair proportions (without donor). Each dot represents one sgRNA. R² and p-value indicate correlation strength; A linear regression line is overlaid in red. M Schematic of Cas9-induced DSB repair outcomes. Without donor: NHEJ and MMEJ; with donor: NHEJ, MMEJ, and HDR (facilitated by MMEJ). E, J Each dot represents one independent experiment (n = 3, > 10 embryos per group). F, K Each dot represents a single blastocyst from three independent experiments. Statistical significance was assessed using unpaired two-sided t-tests (E, J) and two-sided Mann–Whitney U tests (F, K). B, F, K Red bars or dashed lines indicate the mean ± SEM. D, M CRISPR, and DNA icons were created with BioRender. Created in BioRender. Hongyu, C. (2025) https://BioRender.com/qxdvotz. Other elements were designed by the authors. Sample sizes and exact p-value are shown in the figure. Source data are provided as a Source Data file.