In vivo CRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization in Toxoplasma

The research field to identify and characterize virulence genes in Toxoplasma gondii has been dramatically advanced by a series of in vivo CRISPR screens. Although subcellular localizations of thousands of proteins were predicted by the spatial proteomic method called hyperLOPIT, those of more than 1000 proteins remained unassigned and their essentiality in virulence was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned proteins and performed in vivo CRISPR screens. As a result, we identified several in vivo fitness-conferring genes that were previously unreported. We further characterized two candidates, TgGTPase and TgRimM, which are localized in the cytoplasm and the apicoplast, respectively. Both genes are essential for parasite virulence and widely conserved in the phylum Apicomplexa. Collectively, our current study provides a resource for estimating the in vivo essentiality of Toxoplasma proteins with previously unknown localizations. IMPORTANCEToxoplasma gondii is a protozoan parasite that causes severe infection in immunocompromised patients or newborns. Toxoplasma possesses more than 8000 genes; however, the genes that determine parasite virulence were not fully identified. The apicomplexan parasites, including Toxoplasma, developed unique organelles that do not exist in other model organisms; thus, determining the subcellular location of parasite proteins is important for understanding their functions. Here, we used in vivo CRISPR genetic screens that enabled us to investigate hundreds of genes in Toxoplasma during mouse infection. We screened approximately 600 parasite proteins with previously unknown subcellular localizations. We identified many novel genes that confer parasite fitness in mice. Among the top hits, we characterized two essential virulence genes, TgGTPase and TgRimM, which are widely conserved in the phylum Apicomplexa. Our findings will contribute to understanding how apicomplexans adapt to the host environment and cause disease.