Functional Characterization of 11 Tentative Microneme Proteins in Type I RH Strain of Toxoplasma gondii Using the CRISPR-Cas9 System

<i>Toxoplasma gondii</i>, a pathogenic apicomplexan parasite, infects approximately one third of the world's population and poses a serious threat to global public health. Microneme proteins (MICs) secreted by the microneme, an apical secretory organelle of <i>T. gondii</i>, play important roles in the invasion, motility, and intracellular survival of <i>T. gondii</i>. In this study, we selected 11 genes of interest (GOIs) of <i>T. gondii</i>, tentative MICs predicted to be localized in micronemes, and we used the CRISPR-Cas9 system to construct epitope tagging strains and gene knockout strains to explore the localization and function of these 11 tentative MICs. Immunofluorescence assay showed that nine tentative MICs (TGME49_243930, TGME49_200270, TGME49_273320, TGME49_287040, TGME49_261710, TGME49_205680, TGME49_304490, TGME49_245485, and TGME49_224620) were localized or partially localized in the microneme, consistent with the prediction. However, TGME49_272380 and TGME49_243790 showed different localizations from the prediction, being localized in the endoplasmic reticulum and the dense granule, respectively. Further functional characterization of the 11 RHΔ<i>GOI</i> strains revealed that deletion of these 11 GOIs had no significant effect on plaque formation, intracellular replication, egress, invasion ability, and virulence of <i>T. gondii</i>. Although these 11 GOIs are not essential genes for the growth and virulence of tachyzoites of type I RH strain, they may have potential roles in other developmental stages or other genotypes of <i>T. gondii</i>. Thus, further research should be performed to explore the possible role of the nine <i>mic</i>s and the other two GOIs in other life cycle stages and other genotypes of <i>T. gondii</i>.