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

Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are injected into the skin by infected mosquitoes and must reach the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that eventually enter the bloodstream and invade erythrocytes, causing malaria. The sporozoite's journey requires cell traversal, where sporozoites transiently enter and exit host cells, lysing membranes to move deeper into tissue and evade destruction immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish an exoerythrocytic form. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host factors. 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 of HC-04.2B3 and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining. Optimized traversal and invasion assays led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. Disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens confirmed utility of this platform. While no new host factors for malaria were identified in this initial study, we adapted a tractable system for dissecting hepatocyte biology and interactions between liver-tropic pathogens and their host cells, including P. falciparum. This study provides the basis for genome-wide CRISPR screens to uncover hepatocyte biology and host determinants of infection.