Supplementary Figure 8: Appearance of unwound state over time and Cas9–RNA induced unwinding of DNA with roadblocks of single base pair mismatches.
From: Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis
(a) Schematics of smFRET DNA unwinding experiments as described in Fig. 2a. E Histogram of DNA target only (nPD=1) is shown. (b) E histograms of 1 nPD DNA target before and at different time points after adding 100 nM dCas9-HF1-RNA. Rapid appearance of a peak near E=0.5 indicates that Cas9-RNA induced DNA unwinding occurs as soon as Cas9-RNA-DNA complex forms. The number of PAM-distal mismatches (nPD) is shown in cyan. (c) Unwinding of DNA with internal single base mismatches at positions 16th and 18th. E histograms were obtained at [Cas9-RNA] =100 nM or in its absence. Idealized E traces, accompanied with the time traces of donor and acceptor intensities, show transient unwinding observed in a subset of molecules. It is also possible that a significant fraction of such transitions, with lifetime <0.1s, were not detected due to limited time-resolution of these experiments (0.1s). No FRET transitions were observed for DNA only (without Cas9-RNA).
