CRISPR-Cas9 Mediated Disruption of the pfkelch13 Gene and Its Role in Artemisinin Partial Resistance in Nigerian Plasmodium falciparum Isolates

Artemisinin partial resistance in Plasmodium falciparum, driven primarily by mutations in the Kelch13 (pfkelch13) gene, threatens the efficacy of artemisinin-based combination therapies (ACTs) that underpin global malaria treatment guidelines. Surveillance of pfkelch13 mutations in Nigerian P. falciparum populations and functional validation through gene editing tools is urgently needed. This study investigated the functional relevance of pfkelch13 genetic variants detected in clinical P. falciparum isolates from Anambra State, Southeast Nigeria, using CRISPR-Cas9 genome editing in the P. falciparum 3D7 reference strain. Pfkelch13 gene sequences were obtained from 120 patient-derived isolates through PCR and Sanger sequencing. Non-synonymous variants were identified and their predicted structural impacts assessed using protein modeling tools. The most frequently occurring novel variant was introduced into the 3D7 background using CRISPR-Cas9 ribonucleoprotein editing. Ring-stage survival assays (RSA0-3h) were performed on edited parasites and compared against wild-type and validated C580Y-mutant controls. Three novel pfkelch13 variants were identified in the clinical isolate collection. Introduction of the F446I-equivalent variant produced a modest RSA survival increase (3.8% vs 0.4% wild-type), below the validated resistance threshold (RSA greater than 1%). These data suggest that currently circulating pfkelch13 variants in Anambra isolates do not confer full artemisinin resistance, though continued surveillance is recommended. Keywords: Plasmodium falciparum, CRISPR-Cas9, pfkelch13, artemisinin resistance, Anambra State.