Characterization of P. falciparum gametocyte-essential genes using a novel genetic screen

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Plasmodium falciparum, the causative agent of malaria, continues to be a major global burden. Even today, the vast majority of the parasite’s genome has yet to be characterized. This has greatly hindered our ability to develop new antimalarials, therapies, and vaccines. This is particularly true of the sexual gametocyte stage of the parasite, which follows the symptomatic stages in the human host and is transmitted to the mosquito. Gametocytes are not easily cleared by current drug regimens, allowing transmission to occur even after treating an infected individual. To date, there have been few ways to study multiple Plasmodium genes at once, particularly for stages outside of the blood stage. I have worked to develop a new gene editing system that is both accessible and highly scalable. Using Cas9 base-editing, specific C-to-T mutations can be made to introduce early terminations using only a gRNA. This system is highly efficient and can be used to knock out multiple genes in a pooled format. In order to screen for genes essential to gametocyte development, I have generated a list of 250 genes predicted to be essential for the sexual stage based on transcriptomic abundance, proteomic abundance, and evolutionary conservation. I hypothesize that these abundantly expressed and highly conserved genes will be essential to the gametocyte stage, and will incur a fitness cost when silenced. In order to test this, I will perform a genetic screen with this novel editing system using a fluorescent marker for gametocytes. Sorting and sequencing of fluorescent parasites will identify underabundant gRNAs, representative of gametocyte essentiality. The top five hits from our genetic screen will then be individually knocked out and phenotyped to examine the morphology and impairment on mosquito transmission. The findings of this proposal will aid in identifying new drug and vaccine targets that could help prevent the spread of malaria.