Fig. 2
From: Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2
AcrIIC2Nme inhibits sgRNA binding. a Purification of His-tagged Nme1Cas9 + sgRNA co-expressed with AcrIIC2Nme or a type I anti-CRISPR protein (AcrIE2) using Ni-NTA chromatography. Analysis of the resulting elutions included SDS-PAGE followed by Coomassie staining (upper panel) and denaturing polyacrylamide/urea gel followed by SYBR Gold staining (lower panel). b Gel filtration chromatography shows that AcrIIC2Nme interacts with sgRNA-free Nme1Cas9 (upper panel), but fails to bind to the Nme1Cas9-sgRNA complex (lower panel). The Nme1Cas9-sgRNA-AcrIIC2Nme and Nme1Cas9-AcrIIC2Nme-sgRNA samples were reconstituted by incubation of purified Nme1Cas9, sgRNA, and AcrIIC2Nme at a molar ratio of 1:1.3:4 on ice. Each component was added in the order listed, with an intermittent incubation of 30 min before adding the next component. All samples were fractionated on a Superdex 200 increase 10/300, and fractions between 11 and 13 mL were analyzed on SDS-PAGE. c Radiolabeled RNAs were incubated with increasing amounts of Nme1Cas9 in the absence (gray) or presence (purple) of AcrIIC2Nme, and the fraction of protein-bound RNA was determined by nitrocellulose filter binding. Source data are provided as a Source Data file. d DNA cleavage assays with Nme1Cas9 and AcrIIC2Nme. The components and order of addition are noted above each lane. The AcrIIC2Nme mutants were mixed with Nme1Cas9 before adding the sgRNA
