Structural Biology Insights Into The Inhibition Mechanism Of The Type I-F CRISPR-Cas System By AcrIF23

Bacteria and archaea have multiple defense mechanisms against invading phages and foreign nucleic acids,of which CRISPR-Cas is an important adaptive immune system.Phages have also evolved a variety of mechanisms to combat this immune defense system of prokaryotes,and the encoding of anti-CRISPR(Acr)is a very important mechanism.At present,some inhibitory mechanisms of Acr proteins have been found,and they act on different stages of CRISPR-Cas system.However,there are still a large number of mechanisms of Acr proteins that have not been elucidated.The research on them is expected to reveal novel inhibitory mechanisms.CRISPR-Cas system is divided into various types,among which I-F CRISPR-Cas system has the most identified Acr protein.This study gradually explored and revealed the inhibitory mechanism of Acr IF23 protein on I-F CRISPR-Cas system.We solved the structure of Acr IF23 and found that it exhibits a novel way of folding.Acr IF23 is similar to Acr IF3 of the reported mechanism.And they all interact with Cas2/3nucleases in the I-F CRISPR-Cas system.However,unlike Acr IF3,Acr IF23 inhibits DNA cleavage activity of Cas2/3,but does not prevent recruitment of Cas2/3 by cr RNA-guided surveillance complexes(Csy complexes).In contrast,Acr IF3 inhibited the DNA cleavage activity of Cas2/3 weakly,but effectively inhibited the recruitment of Cas2/3 by the cr RNA-guided surveillance complex(Csy complex).Based on the structure of Acr IF23,we mutated the surface residues of Acr IF23,and finally determined the region on the surface of Acr IF23 participating in binding Cas2/3.Taken together,the present study revealed a novel AntiCRISPR-Cas mechanism of Acr IF23,which further illustrated that Acr proteins used a highly diverse inhibitory mechanism to exert their effects.