Fig. 3: Co-inhibition of DNA-PK and Polϴ decreases PEn off-target editing.

a Amplicon-seq and CRISPResso2 analysis at four on-target and 13 off-target sites for the indicated PEn configurations using plasmid co-transfections in HEK293T cells. Editing is shown as percentages of modified reads in each sample. Mismatches to the on-target gRNA sequence are highlighted in red lower-case letters, the PBS region is underlined. b Computed specificity scores for all off-target data shown in (a). Specificity scores are calculated as 1 minus the ratio of editing frequency at the off-target site to that at the on-target site resulting in a range between 0 and 1. Each dot in the graph represents one individual off-target, horizontal lines represent median and 95% confidence intervals of n = 3 biological replicates. Significance levels of specificity scores were calculated with the non-parametric Friedman test (paired, two-tailed). Multiple comparisons were corrected using Dunn’s test. Calculated P-values: PEn vs. uPEn >0.9999 (ns), PEn vs. 1iPEn = 0.7544 (ns), PEn vs. 2iPEn = 0.0008 (***), PEn vs. 2⁺iPEn <0.0001 (****). ns not significant. Drug treatments: 1i = DNA-PKi; 2i = DNA-PKi + PolQi1; 2⁺i = DNA-PKi + PolQi1 + PolQi2. Source data are provided as a Source Data file. PBS Primer Binding Site (PBS), PAM Protospacer Adjacent Motif.