Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni

Abstract

Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. 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 in turn constrains our ability to interrogate the function of essential and conditionally essential genes. This thesis aims to address this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression and thereby investigate gene function. To achieve this, a “dead” cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. The tool was validated through successful repression of the arylsulphatase gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was futher validated against the hippicurase gene hipO, and trialled with an Anhydrotetracycline (ATc) inducible promoter, which demonstrated leaky expression in the absence of ATc. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). Finally, CRISPRi-based repression of the flagella genes fliE and fliQ was attempted to trial targeting of essential genes in C. jejuni. To date, this is the first report of a CRISPR-based interference system demonstrated in C. jejuni.