Studies Into The Molecular Mechanism Of Inhibition Of Type I-F CRISPR-Cas System By AcrIF14

Bacteria have long been invaded by other mobile genetic elements,such as bacteriophages.In order to resist this infection,bacteria have evolved a variety of defense mechanisms,and the CRISPR-Cas system is very important.CRISPR-Cas is a unique immune defense system of prokaryotes such as bacteria.Correspondingly,phages have also evolved various mechanisms to destroy the immune system of bacteria,such as encoding AntiCRISPR(Acr)protein.So far,more than 100 Acr proteins have been reported successively through bioinformatics analysis and functional screening.CRISPR-Cas systems can be divided into different categories and subtypes according to different effector complexes,among which type I-F system is one of the subtypes with the most complex composition of Cas protein effector complexes and the most elaborate mechanism of action.Type I-F Acr proteins usually interact with crRNA-guided effector complexes or Cas2/3 nucleases to antagonize the CRISPR-Cas system.This paper reported the three-dimensional structure of the AcrIF14 and its complex with crRNA-guided surveillance(Csy),and elucidates the molecular mechanism of AcrIF14 inhibiting type I-F CRISPR-Cas systems.This paper found that,in addition to interacting with the Csy complex to prevent the complementary pairing of target DNA and crRNA,AcrIF14 also endows the Csy complex with the ability to interact with non-sequence-specific dsDNA,which is somewhat similar to the inhibition mode of AcrIF9.Further structural and biochemical studies on the Csy-AcrIF14-dsDNA complex found that the PAM recognition region of the Cas8f subunit of the Csy complex and the positively charged region in the N-terminal domain of AcrIF14 are important for the non-sequence-specific dsDNA and Csy-AcrIF14 complex interaction,which is clearly different from the mechanism of AcrIF9.The findings of this paper reveal the universality of non-sequence-specific DNA binding induced by Acr protein,provide an idea for the future study of the mechanism of such Acr protein,and lay a foundation for the development of AcrIF14 as a regulatory tool for type I-F CRISPR-Cas system.