Fig. 4. ssDNA substrate bound in the RuvC catalytic pocket in the Cas12i2^wt–crRNA–DNA ternary complex.

a 5-mer substrate ssDNA is positioned in the cleft formed by the RuvC and Nuc domains. The nucleotide sequence of the ssDNA in the catalytic pocket is assumed based on its electron density. b Schematic interactions of substrate DNA and the RuvC and Nuc domains. Hydrogen bonds and salt bridges are indicated with black arrows. Stacking interactions are shown by blue lines. Two Mg²⁺ ions are shown in magenta spheres. c Mutational analysis of key residues interacting with ssDNA or Mg²⁺. d Magnified view of the coordination of two Mg²⁺ ions with RuvC catalytic residues, the scissile phosphate, and waters. The Mg²⁺ ions are shown as large magenta spheres, and water molecules as small red spheres. The ssDNA substrate is shown in orange, the scissile phosphate in yellow. The distance between the phosphorus atom and nucleophile water is indicated, as well as the distance between two Mg²⁺ ions. e Linear plasmid DNA cleavage assay using different metals. f Sanger-sequencing traces from Cas12i2-cleaved targets show staggered overhangs. The major and minor cleavage sites are indicated by red and orange triangles, respectively (left panel). The non-templated addition of an additional adenine, indicated by a red star, is an artifact of the polymerase used for sequencing (right panel).