Fig. 3

The CATI method demonstrates universality in enhancing HDR efficiency. A A schematic representation of HDR progression and the experimental approaches evaluated in this study. B Assessment of seven methods to improve knock-in efficiency at Actb locus (left) and Dppa3 locus (right). The Tild method is performed as the baseline. TCTS indicates truncated Cas9-targeted sequence; Biotin indicates biotin modification on donors; TSA indicates the addition of TSA (Trichostatin A, HDAC inhibitor); Cas9-CtIP indicates fusion of Cas9 protein with CtIP functional domain; Rad51 OE indicates the overexpression of Rad51 protein; 2-cell injection indicates the injection of regents at the 2-cell stage. Each dot indicates one biological replicate. C The HITI method failed to improve knock-in efficiency at the Actb and Gata6 loci. Each dot indicates one biological replicate. D The DDRNA-based method failed to improve knock-in efficiency at the Actb and H3.3b loci. Each dot indicates one biological replicate. E Assessment of multiple sgRNA strategies to improve knock-in efficiency at the Actb, Gata6, Cdx2, and H3.3 loci. Each dot indicates one biological replicate. F Representative images of Actb genes treated using the CATI + 2C approach. The scale bar represents 100 μm. Each dot corresponds to one biological replicate. G Representative images of Dppa3 genes treated using the CATI + 2C approach. The scale bar represents 100 μm. Each dot corresponds to one biological replicate. H CATI enhances knock-in efficiency at the monkey CDX2 locus. Left: representative images displaying mCherry-positive embryos. Right: a summary of experiments conducted at the CDX2 locus. Paired t-test; n = 3 technical replicates per sample; error bars represent the standard error of the mean (SEM). The scale bar is set at 100 μm. Each dot corresponds to one biological replicate. I CATI enhances the knock-in efficiency at the monkey H3.3 locus. Left: representative images displaying mCherry-positive embryos. Right: a summary of experiments conducted at the H3.3 locus. Paired t-test; n = 3 technical replicates per sample; error bars represent the standard error of the mean (SEM). The scale bar is set at 100 μm. Each dot corresponds to one biological replicate