Cas9-expressing HC-04 hepatocytes facilitate CRISPR-based analysis of Plasmodium falciparum sporozoite-host interactions

Sporozoites of Plasmodium falciparum , the deadliest malaria parasite, are transmitted into the skin by infected mosquitoes and migrate to the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that, eventually, enter the bloodstream and invade erythrocytes, leading to malaria. The parasite journey involves cell traversal, where sporozoites transiently enter and exit host cells beginning in the skin, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish liver-stage infection. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host determinants. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining in HC-04 Cas9+ (clone 2B3) cells. Optimized traversal and invasion assays with HC-04 2B3 cells led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. As proof-of-concept, we performed a small screen involving disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens, confirming utility of this platform. While no new host factors were identified for malaria parasites in this initial study, we have developed a tractable system for genome-wide CRISPR screens to uncover novel hepatocyte biology and host determinants of infection by liver-tropic pathogens.