Method for deletion of variant surface antigen genes at subtelomeric region of Plasmodium falciparum

Plasmodium falciparum expresses variant surface antigens (VSAs), including PfEMP1, RIFIN, and STEVOR, SURFIN on the surface of infected red blood cells. These antigens interact with host receptors on vascular endothelial and immune cells, contributing to both parasite pathogenicity and immune evasion. VSAs are encoded by large multigene families, comprising dozens to hundreds of genes located primarily in heterochromatic regions such as subtelomeric domains, which are notoriously refractory to genetic manipulation. In addition, since P. falciparum parasites undergo antigenic variation by randomly switching VSA expression, it is challenging to use parasites that stably express target VSAs for experimental purposes. As a result, functional characterization of these VSAs has been limited, despite their well-established clinical significance. Here, we present a novel method for targeted deletion of subtelomeric regions in P. falciparum chromosomes by combining the heterochromatin-accessible AsCas12a-UL nuclease with telomere healing. Using this approach, we successfully deleted both subtelomeric regions of chromosome 2. Furthermore, we achieved simultaneous removal of up to seven subtelomeric regions using tandemly arrayed crRNAs, with an efficiency exceeding 85 %. This method provides a powerful tool for generating VSA-null parasites, facilitating precise genetic dissection of individual VSA gene families and their roles in host–parasite interactions.