Establishing CRISPR interference system in Campylobacter jejuni

Abstract S8

Presenter: Ruby Coates (University of Cambridge)

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 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 in turn constrains our ability to interrogate 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 pyogenes CRISPRi system with C. jejuni-derived promoters of pre-determined activities 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 in two C. jejuni strains (81-176 and M1Cam). Further, I have investigated whether sgRNA target location impacts the efficacy of CRISPRi repression by designing sgRNAs that span the length of the astA gene from 5 to 3. I have also assessed whether the endogenous Type-II CRISPR-Cas system in M1Cam impairs the efficacy of the exogenous CRISPRi through the generation of CRISPRi strains using an M1Cam CRISPR-Cas9 deletion mutant. Finally, I have applied the CRISPRi system to target further genes, including the hippicurase enzyme hipO, and a series of motility genes, including flaA, flaB, and flgR. To date, this is the first report of a CRISPR-based interference system demonstrated in C. jejuni.

About the presenter

I am a final year PhD student in Dr. Andrew Grant’s group at the University of Cambridge.

Presenting in Speaking session 4 - Survival and application