Supplementary Figure 3: Off-target binding locations occur at non-random, guide sequence-specific sites.
a GC content or PAM site distribution across the λ-genome. Distribution of canonical PAM sites (NGG) and alternative PAM sites (NAG) across λ-DNA (Top). Distribution of GC content across λ-DNA (Bottom). Bin size = 200 bp. b Average time-binned intensity histograms for λ2, λ4 and nt guide. Off-targets are induced on force stretching DNA to 40 pN and show recurrent non-random off-target binding locations. Arrows marks on-target sites for λ2 (18.5 kb) and λ4 (30.5 kb). c,d,e Histograms (500 nt bin-width) of mapped off-target binding events at 40 pN for 3 guides (λ2-, λ4- and n.t.-crRNA, respectively). Each guide experiment was repeated in triplicate (λ2-crRNA, n = 278, 645 and 311; λ4-crRNA, n = 437, 210 and 150; n.t.-crRNA, n = 605, 876 and 469). f Pearson’s correlation analysis between count normalized histograms of each binding localization repeat (Rep. 1–3) and guide. A strong correlation (dark red) is observed between experiments with the same crRNA guide but not (light red) between different guides, demonstrating that force-induced off-target binding is DNA and crRNA sequence specific. g Kymograph of λ-DNA in the presence of dCas9 with 5′-Cy3-nt-crRNA:tracrRNA (green) and hRPA-eGFP (blue). Off-target binding is observed with increasing force. hRPA binding is observed on nicked DNA and large ssDNA regions. To maintain constant force, extension increases to compensate for DNA nick formation. Off-target Cas9 binding and hRPA binding are mutually exclusive suggesting that two DNA strands are required for Cas9 binding. h Kymograph of 100 nM wtCas9 in complex with non-target 5′-Cy3-nt-crRNA:tracrRNA. Overstretched nicked DNA generates large ssDNA regions, which Cas9 no longer binds. Source data.
