CRISPR-Cas9-Mediated Functional Characterisation of Immune Evasion Genes in Plasmodium falciparum Isolates from Nigeria

Functional characterisation of P. falciparum immune evasion genes, particularly those encoding PfEMP1 and STEVOR proteins, is essential for understanding the molecular basis of severe malaria pathogenesis and for identifying novel therapeutic targets. This doctoral study employed CRISPR-Cas9 genome editing to functionally characterise immune evasion genes in Nigerian P. falciparum clinical isolates. Target genes encoding domain cassettes DC8 and DC13 of PfEMP1, associated with cerebral malaria pathogenicity, were disrupted in three clinical isolates adapted for ex vivo culture. Parasites with and without disruptions were compared for rosetting frequency, cytoadherence to ICAM-1-expressing human brain microvascular endothelial cells, and immune evasion from antibody-mediated growth inhibition. Disruption of DC8-encoding var genes reduced rosetting frequency by 74.3 percent and cytoadherence by 68.7 percent. Antibody-mediated growth inhibition assays demonstrated that DC8-disrupted parasites were significantly more susceptible to immune killing using sera from malaria-immune Nigerian adults. STEVOR protein knockdown reduced merozoite invasion efficiency by 31.4 percent. These functional data, derived from authentic Nigerian clinical isolates, establish DC8 and STEVOR as validated functional virulence factors in locally circulating parasites, providing a rational basis for prioritising these targets in Nigeria-relevant subunit vaccine development efforts. Keywords: CRISPR-Cas9, Plasmodium falciparum, immune evasion, PfEMP1, vaccine target.