| Abstract [eng] |
CRISPR-Cas is a diverse prokaryotic system that provides adaptive immunity against foreign nucleic acids. Bacteriophages have developed a vast variety of strategies to escape CRISPR-Cas protection in an evolutionary bacterial-phage arms race. One of the strategies is phage-encoded small proteins, named anti-CRISPRs (Acrs), that inhibit CRISPR-Cas protection and enable phage evasion. In this work, type I-F CRISPR-Cas from Aggregatibacter actinomycetecomitans D7S-1 bacteria (Aa-CRIRSPR-Cas) was employed to investigate AcrIF6 and AcrIF9 inhibitory effects. In Aa-CRISPR-Cas, the effector complex (Cascade) binds the DNA target and triggers its hydrolysis by Cas2/3 helicase/nuclease. AcrIF6 and AcrIF9 bind to Cascade and hinder DNA target recognition. In addition, AcrIF9 promotes non-specific interactions with DNA, which may facilitate CRISPR-Cas inhibition. Further, the AcrIF9 crystal structure was solved at 2.3 Å resolution and structure-based mutagenesis was employed. Putative AcrIF9 interaction surfaces with DNA and Cascade were identified, following the construction of seven AcrIF9 mutant vectors. The AcrIF9 mutans were expressed and purified. Finally, the significance for Cascade and DNA binding of the predicted interaction surfaces was confirmed by biochemical assays. |
