📖 ABSTRACT/OVERVIEW
Gonorrhoea, caused by Neisseria gonorrhoeae, is a globally important sexually transmitted infection with rapidly expanding antimicrobial resistance threatening treatment options. Vaccine development against N. gonorrhoeae has been hindered by limited understanding of essential virulence genes that are conserved across strains and represent ideal vaccine targets. CRISPRi screens enabling genome-wide transcriptional repression offer a powerful approach to systematically identify essential virulence determinants. This dissertation conducted a genome-wide CRISPRi screen in clinical N. gonorrhoeae isolates from North West Nigeria to identify essential virulence genes as vaccine development candidates. Fifty N. gonorrhoeae isolates were collected from patients with confirmed gonorrhoea attending infectious disease clinics in Kano State. Whole genome sequencing characterised isolate diversity and resistance gene profiles. A sgRNA library targeting all 2,100 annotated N. gonorrhoeae genes was designed and cloned into a dCas9-based CRISPRi vector adapted for N. gonorrhoeae transformation. The library was subjected to competitive growth assays under standard and immune-stress conditions (human serum, neutrophil co-culture). Essential genes were identified by significant sgRNA depletion, and conditionally essential virulence genes were identified by stress-specific depletion. Thirty-eight essential virulence genes not previously characterised in gonorrhoea vaccine literature were identified, including novel outer membrane biogenesis factors. This dissertation proposes a rational vaccine antigen prioritisation framework based on CRISPRi fitness data. Keywords: CRISPRi, Neisseria gonorrhoeae, virulence genes, vaccine development, Kano State
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