A splitCas9 phenotypic screen in Toxoplasma gondii identifies proteins involved in host cell egress and invasion.

Apicomplexan parasites, such as Toxoplasma gondii, have specific adaptations that enable invasion and exit from the host cell. Owing to the phylogenetic distance between apicomplexan parasites and model organisms, comparative genomics has limited capacity to infer gene functions. Further, although CRISPR/Cas9-based screens have assigned roles to some Toxoplasma genes, the functions of encoded proteins have proven difficult to assign. To overcome this problem, we devised a conditional Cas9-system in T. gondii that enables phenotypic screens. Using an indicator strain for F-actin dynamics and apicoplast segregation, we screened 320 genes to identify those required for defined steps in the asexual life cycle. The detailed characterization of two genes identified in our screen, through the generation of conditional knockout parasites using the DiCre-system, revealed that signalling linking factor (SLF) is an integral part of a signalling complex required for early induction of egress, and a novel conoid protein (conoid gliding protein, CGP) functions late during egress and is required for the activation of gliding motility. Establishing different indicator lines and applying our conditional Cas9 screen could enable the identification of genes involved in organellar biogenesis, parasite replication or maintenance of the endosymbiotic organelles in the future.