A Conditional Cas9 System for Stage-Specific Gene Editing in P. falciparum

The malaria parasite has a complex lifecycle involving various host cell environments in both human and mosquito hosts. The parasite must tightly regulate gene expression at each stage in order to adapt to its current environment while continuing development. However, it is challenging to study gene function and regulation of essential genes across the parasite's multi-host lifecycle. Thus, we adapted a recently developed a single-plasmid dimerizable Cre recombinase system for rapamycin-controllable expression of Cas9, allowing for conditional introduction of mutations. We explored rates of gene deletion using varying repair template lengths, showing functionality of donor templates under 250bp for homology-directed repair. As a proof of concept, we conditionally disrupted two uncharacterized genes in blood and gametocyte stages, identifying new stage-specific phenotypes.

As progress towards eliminating malaria has stalled, there is a pressing need for new antimalarials and vaccines. Genes essential to multiple stages of development represent ideal candidates for both antimalarials and vaccines. However, much of the parasite genome remains uncharacterized. Conditional gene perturbation approaches are needed in order to study gene function across the lifecycle. Currently available tools are limited in their ability to perturb genes at the scale required for large screens. We describe a tool that allows for conditional introduction of desired mutations by controlling Cas9 with the DiCre-loxP system. We demonstrate the accessibility of this approach by designing gRNA-donor pairs that can be commercially synthesized. This toolkit provides a scalable system for identifying new drug and vaccine candidates targeting multiple stages of the parasite lifecycle.