Fig. 4.

AcrIIC2_Nme activity is mediated through a large electronegative surface. a AcrIIC2_Nme surface-exposed residues targeted for site-directed mutagenesis are shown on the surface of the protein. The side chain positions at which amino acid substitutions did not affect activity are shown in gray, while the three residues that showed the large decrease in activity when substituted are shown in red. b Representative results of the in vivo phage plaque assay. Serial dilutions of phage Mu plated on E. coli reveal that E17A, E24A, and D108A mutants have lost the ability to inhibit CRISPR-Cas9. c Representative circular dichroism spectroscopy scans of wild type anti-CRISPR (red) and three inactive mutants (E17A, blue; E24A, green; D108A, purple) show that the inactive mutants maintain their secondary structure. d Co-purification of AcrIIC2_Nme mutants with 6x-His-tagged Nme1Cas9 reveals decreased binding of the inactive anti-CRISPRs to Nme1Cas9, and corresponding increase in the amount of Cas9-bound sgRNA. e In vitro DNA cleavage assays with wild type Nme1Cas9 and the site-directed mutants in the bridge helix domain in the presence and absence of AcrIIC2_Nme. f Sequence alignment of the bridge helix of Nme1Cas9 and SpyCas9. The residue numbers of Nme1Cas9 are shown above the sequence. The amino acids that are identical in both Nme1Cas9 and SpyCas9 are shown with yellow background. The Nme1Cas9 amino acids that interact with AcrIIC2_Nme are shown in red. Residues T62 and T73 of SpyCas9 are highlighted by blue stars. g DNA cleavage assay with the wild type or mutant SpyCas9 either in the presence or absence of AcrIIC2_Nme to test whether AcrIIC2_Nme protein inhibits the activity of SpyCas9 mutants