Supplementary Figure 1: No binding is observed with crRNA alone, with crRNA:tracrRNA or crRNA:Cas9 at low or high force or with nt-crRNA:tracrRNA:dCas9 at low force.

a Kymograph of force-stretched λ-DNA in the presence of 5′-Cy3-λ2-crRNA without tracrRNA or dCas9. b Kymograph of force-stretched λ-DNA in the presence of annealed 5′-Cy3-λ2-crRNA:tracrRNA. c Kymograph of force-stretched λ-DNA in the presence of dCas9 and 5’-Cy3-λ2-crRNA without tracrRNA. d Kymograph of low force-stretched (10 pN) λ-DNA in the presence of dCas9 and 5′-Cy3-nt-crRNA:tracrRNA. Vertical scale bar = 1 μm. e Bulk cleavage assay of Cy3-labeled λ2 On-Target DNA (20 nM) (Supplementary Table 1) with λ2-crRNA:tracrRNA:wtCas9 (50 nM). Visualized by 18% denaturing PAGE with separate fluorescent detection for Cy3 and Cy5 fluorescence. Lane 1, no Cas9 complex, Lane 2, unlabeled λ2-crRNA:tracrRNA and wtCas9 (Supplementary Table 1), Lane 3 5′-Cy5-λ2-crRNA:tracrRNA and wtCas9 (Supplementary Table 1). Cy5 labeling of the modified crRNA does not affect λ2 target cleavage efficiency. f Image Analysis Pipeline i) Kymographs are time-binned and their average intensity calculated ii) Bead edge is defined as point where intensity hits background intensity of region known to be DNA iii) Bead trimmed DNA is linearly mapped across known λ-DNA sequence, alignments corrected on the basis of on-target, then smoothed by FFT and intensities within each time window normalized iv) Binding events are detected using ridge detection ImageJ plugin v) Lengths of binding events are calculated and used to build dwell-time histograms. Vi) Position of each event is calculated, mapped back to λ-DNA sequence and used to build histograms of binding sites.