Supplementary Figure 5: Example FRET trajectories for binding to bubble constructs and FRET histograms of crRNA only, double-stranded PAM and ssDNA. | Nature Structural & Molecular Biology

Supplementary Figure 5: Example FRET trajectories for binding to bubble constructs and FRET histograms of crRNA only, double-stranded PAM and ssDNA.

From: DNA stretching induces Cas9 off-target activity

Supplementary Figure 5: Example FRET trajectories for binding to bubble constructs and FRET histograms of crRNA only, double-stranded PAM and ssDNA.

a FRET histograms of the full bubble DNA constructs with 10 nM 5′-Cy5-λ2-crRNA:tracrRNA only (no dCas9). A small population (21%, green, n = 1530) of high FRET molecules bind on-target bubble DNA but not off-targets 1–3 (n = 1610, 2094, and 2120 respectively). b Representative FRET trajectories for λ2-crRNA:tracrRNA:dCas9 binding on-target and off-target bubble constructs. Stable high FRET is observed for on-target and off-targets 1 and 3 followed by single-step photobleaching. For off-target 2 dynamic equilibrium between FRET of ~0.55 and ~0.85 is observed followed by single-step photobleaching. c FRET histograms for ssDNA targets with 10 nM 5′-Cy5-λ2-crRNA:tracrRNA:dCas9. A small population (25%, green, n = 3717) of high FRET molecules bind the on-target constructs but not off-targets 1–3 (n = 2234, n = 1499, n = 1012 for OT1, OT2 and OT3 respectively). d FRET histograms for double-stranded PAM constructs with 10 nM 5′-Cy5-λ2-crRNA:tracrRNA:dCas9 show a small high FRET population (20%, n = 1976; 22%, n = 3062; 23%, n = 2127; 31%, n = 2874; for ON, OT1, OT2 and OT3, respectively). e FRET histograms for a small (6 nt) PAM-proximal bubble. A bound population is observed for on-target (FRET = 0.97 ± 0.02, 36%, n = 3863), OT1 (FRET = 0.23 ± 0.02, 52%, n = 2466) and OT2 (FRET = 0.27 ± 0.02, 75%, n = 3704) but not for OT3 (n = 626).

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