Fig. 2. Protein contacts that open and kink the nontarget DNA strand.

(A) Structural comparison of sgRNA-DNA complexes in existing DNA-bound Cas9 structures (with sgRNA scaffold omitted for clarity). The ordered nontarget DNA strand in the dsDNA-bound structure is shown with a simulated-annealing omit F_o-F_c electron density map contoured at 1.5 σ. The sgRNA-DNA complexes in the ssDNA-bound (PDB ID 4OO8) and PAM-containing partial duplex-bound (PDB ID 4UN3) structures are colored in cyan and beige, respectively. (B) Schematic showing key Cas9-dsDNA interactions. For clarity, only the unwound nontarget strand and the seed RNA–target DNA heteroduplex are shown. Residues specifically interacting with the PAM motif are highlighted in red. Hydrogen bonds or electrostatic interactions are indicated by dashed lines; hydrophobic contacts and van der Waals interactions are shown as solid lines. (C) Position of nontarget strand in RuvC active site. The red sphere depicts the scissile phosphate. Notably, the RuvC domain in the dsDNA-bound structure (marine) superimposes well with that in the Mn²⁺-bound apo-Cas9 form (gray), with two Mn²⁺ ions fit snugly in the active center.