Functional Characterization of Eight Zinc Finger Motif-Containing Proteins in Toxoplasma gondii Type I RH Strain Using the CRISPR-Cas9 System

The Zinc finger protein (ZFP) family is widely distributed in eukaryotes and interacts with DNA, RNA, and various proteins to participate in many molecular processes. In the present study, the biological functions of eight ZFP genes in the lytic cycle and the pathogenicity of Toxoplasma gondii were examined using the CRISPR-Cas9 system. Immunofluorescence showed that four ZFPs (RH248270-HA, RH255310-HA, RH309200-HA, and RH236640-HA) were localized in the cytoplasm, and one ZFP (RH273150-HA) was located in the nucleus, while the expression level of RH285190-HA, RH260870-HA, and RH248450-HA was undetectable. No significant differences were detected between seven RHΔzfp strains (RHΔ285190, RHΔ248270, RHΔ260870, RHΔ255310, RHΔ309200, RHΔ248450, and RHΔ236640) and the wild-type (WT) strain in the T. gondii lytic cycle, including plaque formation, invasion, intracellular replication, and egress, as well as in vitro virulence (p > 0.05). However, the RHΔ273150 strain exhibited significantly lower replication efficiency compared to the other seven RHΔzfp strains and the WT strain, while in vivo virulence in mice was not significantly affected. Comparative expression analysis of the eight zfp genes indicates that certain genes may have essential functions in the sexual reproductive stage of T. gondii. Taken together, these findings expand our current understanding of the roles of ZFPs in T. gondii.