Structure and Mechanism Investigation of anti-CRISPR AcrIIA1 and AcrIIA5 Proteins

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) genes constitute a prokaryotic adaptive immune system against invading phages and plasmids.To counteract this defense mechanism, phages evolved Anti-CRISPR (Acr) proteins that can inactivate the CRISPR-Cas systems.A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems.AcrIIA1 and AcrIIA5, encoded by Listeria monocytogenes prophages and Streptococcus thermophilus, are the most prevalent among the Acr proteins targeting type II-A CRISPR-Cas systems (AcrIIA1) and potently inhibits diverse type II-A and type II-C Cas9 homologs (AcrIIA5), respectively.Here, I investigated the structural and II functions of the anti-CRISPRs proteins.The AcrIIA1 structure displays its dimeric assembly with a novel two-domain architecture.AcrIIA1 exhibits structural similarity to transcriptional factors in the N-terminal domains.When overexpressed in Escherichia coli, AcrIIA1 associates with RNAs, suggesting that AcrIIA1 functions via nucleic acid recognition.AcrIIA5 reveals a novel α/β fold connected to an intrinsically disordered region (IDR).AcrIIA5 directly interacts with single-guide RNA (sgRNA)-Cas9 complex, and hinders proper association between Cas9 and sgRNA required for the nuclease activity of Cas9.Taken together, the results reveals unique structural and functional features of AcrIIA1 and AcrIIA5, suggesting its distinct mode of action and the diversity of the inhibitory mechanisms employed by Acr proteins.