Generation of a Plasmodium falciparum reporter line for studies of parasite biology throughout the life cycle

Abstract

Fluorescence 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 generate PfOBC13GFP, a novel selection marker-free reporter parasite in the background of the African NF54 Plasmodium falciparum line. As docking site, we selected the OBC13 locus that is dispensable for asexual and sexual development in vitro. PfOBC13GFP parasites encode GFP flanked by hsp70 UTRs 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, PfOBC13GFP parasites showed normal development during blood and mosquito stages, and they efficiently infected the main African vector Anopheles coluzzii, overcoming one of the limitations of the previously developed fluorescent reporter lines based on the Pfs47 locus. PfOBC13GFP constitutes a potent tool enabling host-pathogen studies throughout Plasmodium life 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 of PfOBC13GFP, a new fluorescent parasite strain of the human malaria parasite P. falciparum. PfOBC13GFP parasites 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 mosquito vector 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.