CRISPR-Cas9 Mediated Elucidation of Metabolic Enzyme Functions in Plasmodium falciparum for Drug Target Identification

Identifying and validating new drug targets in Plasmodium falciparum is an urgent global health priority given expanding antimalarial drug resistance, and CRISPR-Cas9 gene editing in the malaria parasite provides a powerful tool for functional genomic elucidation of essential metabolic enzymes as drug targets. This dissertation employs CRISPR-Cas9 mediated gene editing to elucidate the functions of three metabolic enzymes in P. falciparum, selected from a computational target priority analysis of the Nigerian P. falciparum genome dataset: phosphoenolpyruvate carboxylase, plasmepsin IX, and the novel pantothenate kinase isoform. Target selection was informed by metabolic flux modelling of the P. falciparum central carbon metabolism and by predicted essentiality scoring from available genome-wide fitness data. Conditional knock-down and complete knockout parasite lines were generated using CRISPR-Cas9 vectors adapted for P. falciparum codon usage, with glmS ribozyme conditional regulation for essential gene targets. Phenotypic consequences of gene disruption were characterised by growth assays across the intraerythrocytic developmental cycle, morphological analysis by electron microscopy, and comparative metabolomics by LC-MS/MS of disrupted versus wild-type parasites. Biochemical enzyme characterisation of recombinantly expressed target proteins enabled inhibitor screening by fluorescence-based enzymatic assays against a repurposing compound library. CRISPR disruption of phosphoenolpyruvate carboxylase severely impaired late trophozoite development, confirming essentiality. Metabolomics of disrupted parasites revealed unexpected compensatory flux through the glyoxylate pathway, providing new mechanistic insights. Inhibitor screening identified three compounds with IC50 values below 1 micromolar against recombinant plasmepsin IX. The dissertation makes original contributions to P. falciparum functional genomics and antimalarial drug target biochemistry. Keywords: CRISPR-Cas9, Plasmodium falciparum, drug targets, metabolic enzymes, antimalarial.