A versatile, marker-free platform for life cycle-wide imaging in Plasmodium falciparum via integration of an exogenous gene cassette into a conserved intergenic locus

The development of a transgenic Plasmodium falciparum line that exhibits robust fluorescence throughout its life cycle in cultured cells and mosquito hosts is valuable for live imaging, tissue localization studies, and quantitative evaluations of parasite infectivity. In this study, we utilized Plasmodium-optimized genome editing to integrate an mCherry expression cassette into a specific intergenic locus without gene disruption; thereby successfully minimizing the physiological impact of exogenous gene introduction. The resulting transgenic parasite line, NF54-mCh, exhibited intense fluorescence across all developmental stages; including intraerythrocytic asexual and sexual blood stages; mosquito ookinete, oocyst, and sporozoite stages; and liver stages. Cultured NF54-mCh exhibited normal intraerythrocytic proliferation, gametocytogenesis, and gametocyte maturation, with efficient transmission to mosquito vectors. The marker brightness in salivary gland sporozoites allowed for visual, non-invasive identification of infected mosquitoes, thus facilitating dissection. Sporozoites dissected from salivary glands were highly infectious to livers in humanized mice; and allowed completion of the full life cycle, as evidenced by the appearance of ring-stage parasites in inoculated human erythrocytes. NF54-mCh serves as a parental line for performing additional genetic modifications, because the CRISPR/Cas9-based genome editing method is free of introduced drug resistance markers. The broader applicability of this strategy was validated by generating fluorescence reporter lines for use in the rodent malaria parasite model systems, Plasmodium berghei and Plasmodium yoelii. In summary, NF54-mCh represents a unique, versatile platform that will accelerate fundamental research and support the future development of innovative malaria control strategies, including studies on new vaccines and drug efficacy. IMPORTANCEA detailed understanding of the molecular and cellular biology of the human malaria parasite Plasmodium falciparum, particularly during infection processes, is essential for efforts in malaria control and elimination. In this study, we generated a marker-free transgenic P. falciparum line which exhibits constitutive, robust fluorescence throughout its developmental stages in erythrocytes, mosquitoes, and hepatocytes, without impairing parasite growth or fitness. The fluorescence marker gene was inserted into an intergenic locus and thus no genes were disrupted. The pfs47 gene is intact, which allows the parasites to evade the mosquito innate system; therefore, the parasite retains the ability for pfs47-dependent infection of refractory Anopheles strains. The transgenic parasites do not contain drug resistance markers, and thus there are no limitations for further genetic manipulation. This transgenic parasite line provides a powerful tool for the study of fundamental parasite infection mechanisms and for the molecular analysis of host-parasite interactions.