Fig. 1.

AcrIIC2_Nme inhibits Cas9 activity through an interaction with the bridge helix. a Plaquing of E. coli phage Mu targeted by type II-C Cas9 proteins (Nme1Cas9, HpaCas9, GeoCas9, CjeCas9) in the presence of AcrIIC2_Nme, a type II-C anti-CRISPR with broad activity (AcrIIC1_Nme) and a type I anti-CRISPR (AcrIE2). The sequence identity of the Cas9 proteins as compared to Nme1Cas9 is noted to the right of the figure. b Untagged anti-CRISPR was co-purified with 6x-His-tagged full-length HpaCas9, or domains thereof (c) using Ni-NTA affinity chromatography and the bound proteins were analyzed by SDS-PAGE and visualized using Coomassie staining. c Schematics of HpaCas9 truncations used to identify the domain with which AcrIIC2_Nme interacts. The bridge helix is denoted in dark gray, and the three sequence regions that comprise the RuvC domain are denoted as I, II, and III