Biochemical Study of CRISPR-associated and anti-CRISPR proteins

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR associated (Cas) proteins provide an adaptive immune system of bacteria and archaea against invading foreign nucleic acids. In type I-F CRISPR-Cas system, Cas proteins (Csy1-4) form a surveillance complex with CRISPR RNA (crRNA) to recognize the target nucleic acids. In this study, I report the biochemical characterization of Csy1-Csy2 heterodimer from Xanthomonas albilineans (XaCsy1-Csy2 heterodimer) by analyzing its interaction with crRNA and AcrF2, an anti-CRISPR (Acr) protein from a phage infecting Pseudomonas aeruginosa. Electrophoretic mobility shift assays revealed that the binding of XaCsy1-Csy2 heterodimer to the 5ʹ-handle of the crRNA is sequence-specific. Size exclusion chromatography and isothermal titration calorimetry analyses demonstrated tight binding between the AcrF2 and XaCsy1-Csy2 heterodimer. Furthermore, the X-ray crystal structure of AcrF2 was solved to a resolution 1.34 Å and enabled a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. These results provide biochemical information of the Csy1-Csy2 heterodimer from a previously uncharacterized bacterial species and also suggest that the AcrF2 protein has broad specificity in inhibiting the type I-F CRISPR-Cas system.