Putative gene regulatory functions of Csy1, a component of the CRISPR-Cas adaptive immune system, in the opportunistic pathogen Pseudomonas aeruginosa

It is becoming increasingly clear that clustered regularly interspaced short palindromic repeats; CRISPR-associated (CRISPR-Cas) systems function beyond adaptive immunity. Preliminary data show that CRISPR-Cas deletion from Pseudomonas aeruginosa reduces both in vitro fitness and in vivo virulence. These phenotypes are caused by Csy1, a Cas protein thought to be involved in sequence-specific binding of the 5'-handle of crRNA. We hypothesise that Csy1 regulates expression of genes carrying sequence motifs similar to the 5'-handle. 

We will test this hypothesis using transcriptome analysis of WT and Csy1 deletion mutants, RIP-seq analysis of RNA co-purifying with Csy1 and EMSA to measure sequence specificity of Csy1-RNA interactions. This project will reveal whether Csy1 of the P. aeruginosa CRISPR-Cas system has a gene regulation function and will identify specific RNA motifs targeted by Csy1.

If successful, the data from this seed project will serve as a basis for a larger proposal aimed at validating the Csy1-motif interactions in vivo, generalising Csy1-mediated gene regulation across P. aeruginosa strains and testing the impact of Csy1 on P. aeruginosa virulence in other, more relevant, infection models, such as cell culture and thermally-injured mouse infection models.