Fig. 1. Crystal structure of SpyCas9 reveals an open bilobed architecture and nucleic acid binding clefts.

(A) Cartoon schematic of the polypeptide sequence and domain organization for the type II-A Cas9 protein from S. pyogenes (SpyCas9). Cas9 is predicted to contain a single HNH nuclease domain and a single RuvC nuclease domain. The RuvC domain is made up of three discontinuous segments (RuvC-I to RuvC-III), with the α-helical lobe inserted between the first and the second segments, and the HNH domain inserted between the second and the third segments. Arg, arginine-rich region; Topo, Topo-homology domain; CTD, C-terminal domain. (B) Orthogonal views of the overall structure of SpyCas9 shown in ribbon representation. Individual Cas9 domains are colored according to the scheme in (A). SpyCas9 consists of a nuclease domain lobe and an α-helical lobe. Disordered segments of the polypeptide chain are denoted with dotted lines. (C) Surface representation of SpyCas9 depicting the two nucleic acid binding clefts on the molecular surface. (D) Surface electrostatic potential map of SpyCas9 colored from −10 kT/e (red) to +10 kT/e (blue) (61). (E) Surface representation of SpyCas9 colored according to evolutionary conservation. The representation was generated using the ConSurf server (62) based on the multiple sequence alignment of type II-A Cas9 proteins shown in fig. S1. A disordered segment (residues 571^Spy to 586^Spy, indicated with a black dashed line) covers the apparently conserved patch on the reverse convex surface of SpyCas9.