Fig. 4

The CATI method enhances the efficiency of nucleotide replacement. A Experimental flow diagram illustrating the steps involved. Following ssODN-mediated integration of the EcoRI site, we conducted restriction enzyme site analysis and Sanger sequencing tests. The light symbol represents CRISPR/Cas9-mediated DSBs, while blue lines indicate the EcoRI site. B Distribution of HDR efficiency percentages for the Oct4 and Ctcf loci using baseline and CATI methods. Red rectangles represent low efficiency (0–30%), white rectangles denote intermediate efficiency (30–60%), and blue rectangles signify high efficiency (60–100%). The “number” refers to the total embryos sequenced and analyzed. C Restriction enzyme site analysis demonstrated an increased HDR efficiency using the CATI method. Gel images (left) and quantitative analysis (right) reveal that the CATI approach is more effective than the baseline method at the Oct4 and Ctcf loci. A two-sided Student’s t-test was employed for statistical analysis. Data are presented as mean ± standard error of the mean (s.e.m.). D Comparison of the positive rate in mouse offspring for G93A and A4V mutation mimicry using both baseline and CATI methods. E Distribution of HDR efficiency percentages for G93A and A4V mutations at Sod1 locus using baseline and CATI methods. Pink rectangles represent low efficiency (0–30%), green rectangles denote intermediate efficiency (30–60%), and yellow rectangles signify high efficiency (60–100%). The number refers to the total number of embryos sequenced and analyzed. F Proportion of indel and MMEJ events across the four tested loci. Blue rectangles represent low efficiency (0–30%), green rectangles represent middle efficiency (30–60%), and yellow rectangles represent high efficiency (60–100%). The number refers to the total number of embryos or mice sequenced and analyzed. G Schematic diagram of CATI strategy enhancing gene knock-in efficiency