Toxoplasma gondii forms a parasitophorous vacuole (PV) upon invading a cell. The host adopts a cell-autonomous response driven by interferon-gamma (IFN-γ), in which interferon-inducible proteins lead to parasite growth inhibition and clearance within the vacuole. Highly virulent strains of T. gondii are known to evade this anti-T. gondii IFN-γ-dependent host defense; however, the molecular mechanisms are incompletely understood. Through in vivo CRISPR/Cas9 screening, we identified candidate genes associated with T. gondii virulence and resistance to the IFN-γ-dependent host defense. Among these candidates, we selected genes encoding non-secreted proteins linked to fundamental cellular processes; apart from rhoptry and dense granule proteins, to assess their influence on virulence. Notably, parasites lacking the deubiquitinase txDUB1 or DNA repair protein 1(txDRP1) exhibited significantly lower virulence than wild-type parasites infecting in wild-type mice, while being comparable to wild-type parasites infecting in mice lacking the IFN-γ receptor. Collectively, these data suggest that txDUB1 and txDRP1 are important for resistance to IFN-γ-dependent anti-T. gondii host defense. We aim to further explore their molecular functions in detail.