Advancing Functional Genomics in P. falciparum Through Systems Biology Approaches

Abstract

Plasmodium falciparum, the parasite responsible for the deadliest form of malaria, has a complex lifecycle involving various host cell environments in both human and mosquito hosts. The parasite must tightly regulate the expression of each of its ~5500 genes at each stage in order to adapt to its current environment while continuing development. Roughly half of these genes are essential to the asexual blood stage, making it challenging to study gene function and regulation in subsequent stages. Thus, I established a multi-stage transcriptomic dataset that encompasses the complete lifecycle of the parasite. This allows for the comparison of genes across each of the lifecycle stages, helping to generate new hypotheses. To better validate stage-specific phenotypes, I adapted a dimerizable Cre recombinase system using loxP-flanked stop elements for leak-free controllable over-expression and Cas9-directed knockouts. Using a high-fidelity Cas9 under this inducible system, we were able to conditionally perform genome editing by homology-directed repair, editing at specific timepoints and lifecycle stages. I further improved the versatility of this system by establishing a scalable cloning approach for pairing synthesized gRNA-donor sequences. Expanding this system to recently developed Plasmodium CRISPRa and CRISPRi systems, stable and scalable CRISPR screens can be generated for over- and under-expression of numerous genes. I generated two conditional knockouts of uncharacterized genes, identifying blood and gametocyte phenotypes. I then explored the ability to screen for transcriptional regulators of parasite development and artemisinin resistance using small-scale CRISPRa and CRISPRi screens. I envision these novel approaches will aid in further characterizing the parasite genome across its lifecycle, and ultimately help in developing new antimalarials and vaccines.