a, Scatterplot of PTC tolerance scores in nej1∆ cells calculated independently for each replicate experiment (n = 3132 independently targeted PTCs that were observed in both replicates, Pearson's r = 0.6, p < 2 × 10-16). b, Scatterplot of gene PTC tolerance scores calculated independently for each replicate experiment (n = 1140 independently targeted genes, Pearson's r = 0.7, p < 2 × 10-16). c, Repair templates introducing essential gene PTCs were less tolerated than repair templates introducing dubious ORF PTCs. The experiment included 72 cases of pairs of edit-directing plasmids that both used the same gRNA. In each pair, one plasmid introduced a PTC in an essential gene, and the other introduced a PTC in a dubious ORF overlapping the same essential gene; the latter had the effect of introducing a synonymous or nonsynonymous substitution in the essential gene. Lines correspond to effects of the same gRNA targeting either a PTC to an essential gene or an edit to an overlapping dubious ORF. The edit-directing plasmids targeting PTCs to essential genes were less tolerated than their partners targeting PTCs to the overlapping dubious ORFs (Student's two-tailed paired t-test n = 72, t = 6.5, P = 8 × 10−9). The outer boxplots show the distribution of edit tolerance scores. The centerline of each box corresponds to the data's median value; the top and bottom of the box span from the first quartile to the third quartile of the data; and the whiskers reach to either the data's most extreme values or 1.5 times the interquartile range.