AcrIIA8 is a putative phage structural protein of the HTJ2 family that does not inhibit Streptococcus pyogenes Cas9

Anti-CRISPR (Acr) proteins are phage-encoded anti-defense factors that suppress CRISPR-Cas immunity in bacteria. AcrIIA8 was previously identified as an inhibitor of Streptococcus pyogenes Cas9 (SpyCas9) through functional assays of metagenomic libraries. Here, we report that AcrIIA8 does not inhibit SpyCas9 in biochemical assays under a range of buffer conditions and temperatures. The solution structure and dynamics of AcrIIA8 reveal a six-stranded β-barrel fold with flexible β1-β2 and β2-β3 loops, characteristic of phage virion-assembly proteins. In addition, genomic context analysis places AcrIIA8 and its homologs within conserved prophage morphogenetic regions at the position expected for type II head-tail joining (HTJ2) proteins. We further detected no interaction between AcrIIA8 and SpyCas9 in NMR titration experiments, suggesting that they do not specifically associate. Taken together, these findings argue against assigning AcrIIA8 as a SpyCas9 inhibitor and instead support its annotation as a putative phage structural protein of the HTJ2 family.