Development of a CRISPR interference system in Campylobacter jejuni

Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuniresponsible for 80% of these infections. There is no current vaccine and antibiotic resistance is emerging. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which impacts studies into the function of essential and conditionally essential genes. My project aims to address this by applying a CRISPR-based interference system known as CRISPRi in C. jejuni as a means to control gene expression and thereby investigate gene function. To validate the CRISPRi system in C. jejuni, I have paired the dCas9 and sgRNA backbone from the Streptococcus pyogenesCRISPRi system with several C. jejuni-derived promoters to develop a series of CRISPRi constructs targeting several genes. Through rigorous sgRNA target design I have successfully targeted and repressed expression of the endogenous arylsulphatase (AstA) enzyme, as well as achieving partial repression of expression of the regulatory flagellar protein FlgR in two clinically relevant C. jejuni strains. This is the first report of a CRISPRi system for Campylobacter.