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

Infection with the apicomplexan protozoan parasite 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 pathogenesis. However, the functions of some GRAs remain undefined. In this study, we used CRISPR-Cas9 technique to disrupt 17 genes (, and ) in the virulent RH strain. The CRISPR-Cas9 constructs abolished the expression of the 17 genes. Functional characterization of single Δ mutants was achieved using cell-based plaque assay and egress assay, and in BALB/c mice. Targeted deletion of these 17 genes had no significant effect neither on the 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 genes suggest that 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 or virulence in mice, they may have roles in other strains or parasite stages, which warrants further investigations.