Fig. 2. Interactions of dCas9/sgRNA with a PAM-distal mismatched DNA target.
(A to F) Representative traces of forward and reverse DNA unzipping with the D15-20mm (A, B, and C) and the D11-20mm templates (D, E, and F). The forward and reverse unzipping forces versus the number of base pairs are presented in black and blue, respectively. The naked DNA unzipping signatures are also presented for comparison (gray). Red arrows indicate the force peaks. The forward unzipping forces populated two signatures: one peak and dual peaks. (G) Positions and forces of the three interactions between Cas9/sgRNA and mismatched DNA: pre-, post-, and intermittent interaction. The hollow triangle indicates the transient interaction, and the black and blue triangles indicate the pre- and post-PAM interactions, respectively. (H) Fractions of dCas9-bound DNA at the expected position with fully matched (target) and mismatched DNAs (D15-20mm and D11-20mm), as revealed by forward unzipping.