Inhibition Mechanisms Of Nuclease Recruitment Of Host Type I-F CRISPR-Cas System By Phage Proteins

CRISPR array and CRISPR-associated genes on bacterial and archaeal genomes together encode an adaptive immune system that helps hosts defend against invasion by phages,plasmids,and other mobile genetic elements.There are six types of CRISPR-Cas systems for prokaryotes,of which approximately 50% belong to type I,including seven subtypes: I-A,I-B,I-C,I-D,I-E,I-F,and I-G.The extensive distribution range and a large number of types of these systems make them highly significant in enabling the study and exploitation of various and abundant microbial resources.Pseudomonas aeruginosa serves as an excellent model organism for investigating the interactions between phages and type I-F CRISPR systems.The type I-F CRISPR-Cas system encodes a surveillance complex consisting of four types of subunits,Cas5 f,Cas6f,Cas7 f,and Cas8 f,along with a segment of cr RNA,known as the Csy complex.Guided by cr RNA,this complex recognizes and binds to exogenous nucleic acid sequences,subsequently recruiting Cas2/3nucleases to initiate cleavage of DNA.In response to the survival pressure exerted by this prokaryotic immunity,phages evolved a series of Acr proteins to suppress the CRISPR-Cas system,and studies on the functional mechanisms of Acr proteins have yielded natural regulatory tools for gene-editing technology based on CRISPR-Cas systems.This study reveals the mechanism through which Acr IF4 and Acr IF5,two Acr proteins of unknown function,inhibit the I-F type CRISPR-Cas system by suppressing the recruitment of the Cas2/3 nuclease by the Csy-DNA complex.Both Acr IF4 and Acr IF5 are capable of forming a ternary complex with the Csy complex and DNA;however,they exhibit distinct binding mechanisms.Acr IF5 exclusively binds to the Csy-DNA complex,whereas Acr IF4 directly binds to the Csy complex itself.In this work,the structures of Csy-Acr IF4-DNA and Csy-DNA-Acr IF5 were resolved by cryo-electron microscopy.Structural analysis and biochemical studies show that Acr IF4 anchors the Cas8f-HB domain in Csy,which is responsible for recruiting Cas2/3 nucleases,preventing it from rotating and performing its nuclease recruitment function,while Acr IF5 competes for the binding position of the Cas8f-HB domain,preventing it from stabilizing in the Csy complex and performing its nuclease recruitment function.In conclusion,this study enriches the understanding of phage-host interactions and lays the foundation for the development of novel and efficient regulatory tools for CRISPR-Cas gene editing technology.