Generation of aPlasmodium falciparumreporter line for studies of parasite biology throughout the life cycle

Abstract

AbstractFluorescence reporter strains of human malaria parasites are powerful tools to study the interaction of the parasites with both human and mosquito hosts. However, low fluorescence intensity in transmission-relevant parasite stages and the choice of insertion loci that cause parasite developmental defects in the mosquito largely limits usefulness of currently available lines. To overcome these limitations, we used a CRISPR-Cas9-mediated approach to generatePfOBC13GFP, a novel selection marker-free reporter parasite in the background of the African NF54Plasmodium falciparumline. As docking site, we selected theOBC13locus that is dispensable for asexual and sexual developmentin vitro.PfOBC13GFPparasites encode GFP flanked byhsp70UTRs that drive strong fluorescence reporter expression throughout blood and mosquito stages, enabling parasite detection by such high throughput methods as flow cytometry. When compared to the parental line,PfOBC13GFPparasites showed normal development during blood and mosquito stages, and they efficiently infected the main African vectorAnopheles coluzzii,overcoming one of the limitations of the previously developed fluorescent reporter lines based on thePfs47locus.PfOBC13GFPconstitutes a potent tool enabling host-pathogen studies throughoutPlasmodiumlife cycle.ImportanceFluorescence reporter strains have been very useful in malaria research, however, up to date they had limitations in mosquito infectivity and fluorescence intensity. Here we report the generation ofPfOBC13GFP, a new fluorescent parasite strain of the human malaria parasiteP. falciparum.PfOBC13GFPparasites are highly fluorescent throughout the life cycle, making them an ideal tool for the study the parasite progression through blood and mosquito stages. They efficiently infect the African mosquitovector A. coluzzii, allowing the study of this African parasite in its biological background. Moreover, strong parasite fluorescence enables flow cytometry and live microscopy characterization of all parasite stages, especially those involved in transmission.