Fig. 4: enAsCas12a knock-in mice demonstrated high on-target and low off-target editing efficiency.
From: Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering
a, Representative allele frequency plots of the crRNAs-targeted sites for Trp53, Apc, Pten and Rb1 demonstrating the type of gene modification generated in SCC samples. b, Nextera sequencing quantifies percent gene modification for the four targeted genes, Trp53, Apc, Pten and Rb1, in SCC samples. Two-way ANOVA with Å Ãdák’s multiple comparisons test was used to assess significance. Data are shown as mean ± s.e.m. P values are labelled. For all genes, N = 5 biological replicates. c, Schematic demonstrating the process of isolating single clone from primary tumour SCC/LUAD samples and surveying the efficiency of single-cell-level quadruple knockout of the four TSGs. Created in BioRender. Zhou, L. (2025) https://BioRender.com/n05e955. d, Quantification of the number of single clones with simultaneous four TSGs edited. e, Circos plot created with RCircos67 with variant information regarding crCD80 (blue), crH2Ab (green) and CD80-H2Ab1-DKO (red) genome. The outer section composed of three scatter plots (one per group) with wheat colour background, representing the average coverage at each 1 Mb window (blue dot, crCD80; green dot, crH2Ab1; red dot, CD80-H2Ab1-DKO). The inner section is composed of three histogram plots (one per group), representing the variants count by windows of 1 Mb (blue track, crCD80; green track, crH2Ab1; red track, CD80-H2Ab1-DKO). f, Heatmap of percent modification of each predicted off-target site for H2Ab1 crRNA. X-axis is composed of vector, crCD80, crH2Ab1 and CD80-H2Ab1-DKO, each with three biological replicates. Y-axis is composed of the surveyed genomic regions, including one on-target site and ten predicted off-target sites for crH2Ab1. g, Bar plot summarizing the percent modification in predicted off-target sites of H2Ab1 crRNA for vector, crCD80, crH2Ab1 and CD80-H2Ab1-DKO. Data are shown as mean ± s.e.m. N = 3 biological replicates for all groups. h, Heatmap of percent modification of each predicted off-target site for CD80 crRNA. X-axis is composed of vector, crCD80, crH2Ab1 and CD80-H2Ab1-DKO, each with three biological replicates. Y-axis is composed of the surveyed genomic regions, including one on-target site and nine predicted off-target sites for crCD80 (crCD80_6 not included owing to primer synthesis failure). i, Bar plot summarizing the percent modification in predicted off-target sites of CD80 crRNA for vector, crCD80, crH2Ab1 and CD80-H2Ab1-DKO. Data are shown as mean ± s.e.m. N = 3 biological replicates for all groups.
