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Infection with the apicomplexan protozoan parasite  Toxoplasma gondii  is an ongoing public health problem. The parasite's ability to invade and replicate within the host cell is dependent on many effectors, such as dense granule proteins (GRAs) released from the specialized organelle dense granules, into host cells. GRAs have emerged as important determinants of  T. gondii  pathogenesis. However, the functions of some GRAs remain undefined. In this study, we used CRISPR-Cas9 technique to disrupt 17  GRA  genes ( GRA11, GRA12 bis, GRA13, GRA14, GRA20, GRA21, GRA28-31, GRA33-38 , and  GRA40 ) in the virulent  T. gondii  RH strain. The CRISPR-Cas9 constructs abolished the expression of the 17  GRA  genes. Functional characterization of single Δ GRA  mutants was achieved  in vitro  using cell-based plaque assay and egress assay, and  in vivo  in BALB/c mice. Targeted deletion of these 17  GRA  genes had no significant effect neither on the  in vitro  growth and egress of the mutant strains from the host cells nor on the parasite virulence in the mouse model of infection. Comparative analysis of the transcriptomics data of the 17  GRA  genes suggest that  GRAs  may serve different functions in different genotypes and life cycle stages of the parasite. In sum, although these 17 GRAs might not be essential for RH strain growth  in vitro  or virulence in mice, they may have roles in other strains or parasite stages, which warrants further investigations.

Functional Characterization of Dense Granule Proteins in Toxoplasma gondii RH Strain Using CRISPR-Cas9 System