The Interaction Between CRISPR-Cas And DNA Phosphorothioation System

In the microbe world,bacteria and archaea are constantly threatened by mobile gene elements such as viruses or plasmids,so they have evolved a variety of defense strategies to protect their genetic stability.The newly found DNA phosphorothioation system belongs to native defense system which consists of the modification protein encoded by dnd ABCDE and the restriction protein encoded by dnd FGH or pbe ABCD downstream.In contrast,the CRISPR-Cas system plays a defense role by integrating foreign nucleic acid sequence into its own genome,thus being called the acquired immune system.Through bioinformatics analysis,both systems are widely distributed among various bacteria and archaea,providing protection for their hosts.And in some bacterial genomes,there exist gene clusters encoding both systems.So,this article set out to answer if there are functional interaction behind the coexistence.In the genome of Salmonella enterica serovar Cerro 87 used in this study,we found a Dnd system composed of dnd BCDE-dnd FGH and type I-E CRISPR-Cas system which was proved inactive in the plasmid transformation assay.We instead adopted well-studied Streptococcus pyogenes typeⅡ-A CRISPR-Cas system.Through the efficiency of plaque（EOP）assay,we found that the Dnd system alone can reduce the phage infection by 10³-fold,while the defense ability of CRISPR-Cas system varies with its spacer(10^2-6-fold).Two systems are compatible in one host and improve the overall resistance by up to 10⁶-fold compared to either system alone.On the other hand,in Cas9 in vitro cleavage assay,we designed three kinds of DNA substrates with different phosphorothioation modification state,and under the same conditions,treated with Cas9 protein.We found that the modified DNA does not significantly inhibit the cleavage activity of Cas9,suggesting that the dual system collaboration is probably because Dnd system-escaped phage will be recognized and cleaved by a CRISPR-Cas system.This study focused on the interaction between the Dnd and CRISPR-Cas system,break the cognition of the independent function of two defense systems,showed the diversity and complexity of the bacterial defense system,and also reflect the coevolution theory between bacteria and viruses.