Reversible Regulation Of Cas12a Activities By AcrVA5-mediated Acetylation And CobB-mediated Deacetylation

The CRISPR system is an adaptive immune mechanism used by bacteria to defend against foreign phage invasion.The Cas12 a protein uses CRISPR RNA(cr RNA)to guide and specifically target the invading foreign gene fragments and cut the fragments so that the bacteria will not be infected.Due to its programmability,like Cas9,Cas12 a has also been developed as a gene editing tool for multiple species.In order to reduce the off-target rate of gene editing,people have developed a variety of methods,including anti-CRISPR proteins,to precisely regulate the intensity and duration of the Cas protein,with a view to developing safer,more efficient,and more precise gene editing tools.Among them,AcrVA5 as an acetyltransferase is an anti-Cas12 a protein,which can acetylate and inactivate Cas12 a protein.This subject further proves that the acetylated modified receptor of AcrVA5 has a wide range of acetylation and can inactivate a variety of Cas12 a proteins.Purification of AcrVA5 and five Cas12 a proteins in vitro proved that AcrVA5 can reduce the cis and trans cleavage activities of Lb Cas12 a,Mb2Cas12a,As Cas12 a,Fn Cas12 a,and Lb5Cas12 a proteins to target double-stranded DNA.In addition,it was also proved that AcrVA5 has broad-spectrum acetyltransferase activity,and CobB protein can deacetylate and modify most of the proteins inactivated by AcrVA5,such as Lb Cas12 a,Mb2Cas12a,As Cas12 a and Fn Cas12 a,and restore their cis and trans cleavage.active.However,CobB protein cannot recover Lb5Cas12 a that is inactivated by AcrVA5,and its mechanism remains to be exploredPrevious studies reported that AcrVA5 modifies the Lys613 site of Lb Cas12 a through acetylation,so that it loses its ability to recognize PAM sites after acetylation.However,this project found that the mutation of Lys613 of Lb Cas12 a to arginine can still be inactivated by AcrVA5-mediated acetylation modification.Therefore,it is speculated that the PAM sequence region contains more than one key amino acid site involved in the recognition of the target DNA by the Cas protein.Through the study of AcrVA5 and CobB protein reversible modification of Cas12 a,this project deepens the understanding of the mechanism of CRISPR and anti-CRISPR as well as the co-evolution mechanism of bacteria and phage,and helps to develop better gene editing tools in the future.