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

Area of Science:

• * Parasitology and molecular biology
• * Genetics and genomics of Plasmodium falciparum

Background:

• * Malaria elimination efforts are stalled, necessitating novel antimalarials and vaccines.
• * Essential genes across the parasite lifecycle are key targets, but much of the genome remains uncharacterized.
• * Studying essential gene function across the complex Plasmodium lifecycle is challenging.

Purpose of the Study:

• * To develop a scalable and conditional gene perturbation system for the malaria parasite.
• * To enable the study of essential genes across multiple host cell environments.
• * To facilitate the identification of new drug and vaccine candidates.

Main Methods:

• * Adaptation of a single-plasmid dimerizable Cre recombinase system for rapamycin-controllable Cas9 expression.
• * Exploration of homology-directed repair efficiency with varying repair template lengths.
• * Conditional disruption of uncharacterized genes in blood and gametocyte stages.

Main Results:

• * Demonstrated functionality of donor templates under 250bp for homology-directed repair.
• * Successfully conditionally disrupted two uncharacterized genes.
• * Identified novel stage-specific phenotypes for the disrupted genes.

Conclusions:

• * The developed toolkit provides a scalable system for conditional gene perturbation in the malaria parasite.
• * This approach facilitates the identification of essential genes for antimalarial and vaccine development.
• * The system enables the discovery of new stage-specific phenotypes and therapeutic targets.