Toxoplasma gondii, 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 T. gondii, play important roles in the invasion, motility, and intracellular survival of T. gondii. In this study, we selected 11 genes of interest (GOIs) of T. gondii, 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 RHΔGOI strains revealed that deletion of these 11 GOIs had no significant effect on plaque formation, intracellular replication, egress, invasion ability, and virulence of T. gondii. 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 T. gondii. Thus, further research should be performed to explore the possible role of the nine mics and the other two GOIs in other life cycle stages and other genotypes of T. gondii.

The highly efficient endodyogeny of tachyzoites is a key process driving acute infection by Toxoplasma gondii. The centrocone is a specialized and critical structure for parasite cell division, but the regulatory mechanisms of centrocone proteins in T. gondii remain poorly understood. In this study, we characterized the centrocone protein SMC_N1, which exhibited periodic expression in tachyzoites, peaking during the synthesis phase. Conditional depletion of SMC_N1 was achieved in the type I RH strain and type II cyst-forming PRU strain using the mAID system combined with CRISPR-Cas9. Depletion of SMC_N1 disrupted IMC assembly, endodyogeny and nuclear division, as well as the stable inheritance of the apicoplast and centrosome, resulting in severe defects in intracellular replication and impaired tachyzoite growth. Collectively, these results indicate that SMC_N1 regulates cell division by coordinating organelle inheritance and cytoskeletal dynamics, ensuring proper replication of T. gondii tachyzoites and provide insights into mechanisms controlling parasite proliferation.