CRISPR-Cas9 Mediated Endogenous RNA Targeting in the Foodborne-Pathogen Campylobacter Jejuni

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein Cas9 is a sequence-specific nuclease, which typically cleaves double-stranded target DNA with the help of CRISPR-RNAs (crRNAs) guides and a trans-activating CRISPR RNA (tracrRNA) (1). In a global transcriptome study, we observed that the CRISPR locus of the food-borne pathogen Campylobacter jejuni is one of the most highly transcribed regions in the genome (Dugar 2013 Plos Genetics); however, its potential role beyond genome defense is unclear. Recently, we have uncovered that the Cas9 from Campylobacter jejuni (CjCas9) can also bind and cleave endogenous mRNAs in a crRNA-dependent manner (2). This discovery raises questions on whether Cas9 could play a role in mediating endogenous gene regulation and virulence or whether RNA targeting by CjCas9 is just an off-target effect. To delineate the potential role of endogenous RNA targeting via CRISPR-Cas9, we investigate the regulation of tracrRNA and crRNAs in different strains of C. jejuni and aim to identify conditions that might affect RNA targeting in C. jejuni via CRISPR locus expression. Also, it is not known whether transcript stability and processing of crRNAs and tracrRNA play any role in maintaining crRNA-mRNA interactions. Using transcript stability assays and RNase-deletion mutant strains, we aim to identify factors that control their stability and interactions with Cas9. Using RIP-seq combined with computational analysis, we are investigating endogenous RNA targets and their cleavage by Cas9 in different C. jejuni strains with varying number of crRNAs. Identifying and understanding the functions of the CRISPR locus, its transcribed products and potential target RNAs, will provide insights into the newly discovered world of CRISPR-Cas9 mediated endogenous RNA targeting and its possible role in growth and virulence of C. jejuni.