PMID:28041849

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

AcrIIA2 and AcrIIA4 inhibit an orthologous Cas9 in heterologous hosts

"these data demonstrate the utility of the AcrIIA2 and AcrIIA4 proteins to inhibit the function of an orthologous Cas9 in heterologous hosts"
Breadth across Cas9 orthologues is attributed to a conserved target surface

"Using the orthologous Spy Cas9, it is clear that AcrIIA2 and AcrIIA4 have broad specificity, given that Lmo Cas9 and Spy Cas9 only share 53% sequence identity."
AcrIIA2 may inhibit cleavage as well as DNA binding, since it was more effective in the cleavage assay than in the dCas9 binding assay

"The enhanced efficacy of acrIIA2 in the cleavage-based Cas9 assay relative to the dCas9 based assay suggests that it may inhibit both binding and cleavage to some degree"
The AcrIIA2 sequence used in this work is GenBank AEO04363.1

"AcrIIA2- (AEO04363.1), AcrIIA3- (CBY03209.1) and AcrIIA4- (AEO04689.1) homologous protein sequences were acquired by BLASTp searches"

PMID:30606466

Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race.

AcrIIA2 binds SpyCas9 where the target DNA would bind

"We show that AcrIIA2 binds SpyCas9 at a position similar to the target DNA binding region."
The occluded residues are the PAM recognition residues, so target duplex detection is prevented

"AcrIIA2 interacts with the protospacer adjacent motif (PAM) recognition residues of Cas9, preventing target double-stranded DNA (dsDNA) detection."
Inhibition is DNA mimicry by acidic residues, competitive with target duplex binding, and uses a binding motif distinct from that of AcrIIA4

"phage-encoded AcrIIA2 appears to act as a DNA mimic that blocks subsequent dsDNA binding by virtue of its highly acidic residues, disabling bacterial Cas9 by competing with target dsDNA binding with a binding motif distinct from AcrIIA4"

PMID:28448066

Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein.

AcrIIA2 inhibits both L. monocytogenes Cas9 and S. pyogenes Cas9 in bacteria and in human cells

"Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells."
AcrIIA2 engagement of Cas9 requires the guide RNA

"Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner."