Figure 3. Cascade exhibits non-canonical binding to protospacers with PAM-proximal or PAM-distal segmented mutations.

(A) Schematics of DNA targets in the PAM-proximal mutation series illustrating mutated (white) or unmutated (green) segments (S1-S6) of the protospacer. Mut[S1], Mut[S1-2], Mut[S1-3] and Mut[S1-4] have segments 1, 1-2, 1-3, and 1-4 mutated, respectively. (B) Histograms of the initial FRET upon binding (average of first 1.5 sec of each event) of each PAM-proximal mutant from [A] bearing either an interfering (purple bars, left column) or priming (light blue bars, right column) PAM exhibit peaks (Gaussian fits, black lines) positioned similar to that of the bona fide and Mut[PAM] targets (top row, same as Figures 1F and 2B) at E_O (0.44), E_C (0.65), or E_I (0.84) (dashed black lines). The recorded events are from one field-of-view of the detector. (C) Mean binding dwell time of each PAM-proximal mutant from [A] bearing either an interfering (purple bars) or a priming (light blue bars) PAM (derived from dwell time distributions, see Figure S2A). Error represents std (3 individual data sets). The dwell time of the bona fide target could not be measured accurately due to the photobleaching and thus arbitrarily set 1040 sec to represent the longer characteristic time scale in Figure 1G. (D) Schematics of DNA targets in the PAM-distal mutation series illustrated as in [A]. Mut[S5-6], Mut[S4-6], and Mut[S3-6] have segments 5-6, 4-6, and 3-6 mutated, respectively. (E) Histograms of the initial FRET upon binding of each PAM-distal mutant from [D] displayed in a similar fashion to [B]. (F) Mean binding dwell of each PAM-distal mutant from [D] bearing either an interfering (purple bars) or a priming (light blue bars) PAM (derived from dwell time distributions, see Figure S2A). Error represents std (3 individual data sets). N.D. is “Not Determined”. See also Table S2.