Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.

Abstract

The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single turnover enzyme that displays a stable product state after double-stranded DNA cleavage. Here, we present cryo-EM structures of pre-catalytic, post-catalytic, and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg2+. In the pre-catalytic state, Cas9 adopts the “checkpoint” conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the post-catalytic state involves a dramatic ~34 Å swing of the HNH domain and disorder of the REC2 recognition domain. The post-catalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the pre-catalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and nucleic acids provide new insights into the mechanism of genome editing by Cas9.