CRISPR-Cas9-Mediated Gene Editing of Plasmodium falciparum Drug Resistance Loci: Functional Validation Using Nigerian Clinical Isolates

CRISPR-Cas9 gene editing technology has transformed the functional validation of drug resistance candidate mutations in Plasmodium falciparum, enabling precise allelic exchange that definitively establishes the causal role of specific mutations in determining antimalarial susceptibility phenotypes. This doctoral research applies CRISPR-Cas9 gene editing to functionally validate drug resistance mutations identified in Nigerian P. falciparum clinical isolates. In the genomic discovery phase, whole-genome sequencing of 300 isolates collected from six Nigerian states identified novel non-synonymous mutations in Pfkelch13, Pfcoronin, and Pfubp1 at allele frequencies above 5 percent. Three priority mutations were selected for functional validation based on predicted structural impact on protein function using AlphaFold2 models and evolutionary conservation analysis. CRISPR-Cas9-mediated allelic exchange was performed in the Dd2 reference strain background to introduce each mutation in isolation. Phenotypic characterisation included ring-stage survival assay with dihydroartemisinin, dose-response assays for artemether and lumefantrine, and transcriptomic profiling of the edited lines under drug stress. Evidence from 2020 to 2024 establishes CRISPR-based functional validation as the gold standard for resistance mutation characterisation and has revealed partial artemisinin resistance conferred by Pfcoronin mutations in African isolates. This research provides functional evidence for or against resistance attribution for three novel Nigerian-specific mutations, directly informing WHO criteria for resistance mutation classification. Findings will be published in a top-tier malaria or genetics journal. Keywords: CRISPR-Cas9, Plasmodium falciparum, drug resistance, functional validation, gene editing.