Fig. 4

The impact of off-targets on sgRNA log-fold changes. a Guide counts as a function of the number of single-mismatch alignments. b Combined effect of multiple perfect alignments (on-targets) and single-mismatch (1-mm) alignments on LFCs. c Distribution of cell-specific fitted off-target activity (average log-fold changes) as a function of the number of single-mismatch off-target alignments (solid line: median across cell lines; shaded area: full range of on-target activity across cell lines). d Relationship between cell line-specific on-target toxicity and cell line-specific off-target toxicity. On-target and off-target toxicity were measured as the average LFC for guides with four perfect alignments and four single-mismatch alignments, respectively. e Effect of a single mismatch between spacer and reference genome as a function of the mismatch position. Position 1 represents the position next to the PAM site. f LFCs of guides with 1 perfect alignment stratified by the number of single-mismatch alignments and by spacer location: PAM-proximal (positions 1 to 10) or PAM-distal (position 11 to 20). p values were assessed using two-sample t tests. N.s.: not significant; ∗∗: p<0.01; ∗∗∗: p<0.001