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Type II Toxoplasma gondii KU80 knockouts (Δ ku80 ) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δ ku80 Δ hxgprt strain measured at the orotate ( OPRT ) and the uracil ( UPRT ) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δ ku80 Δ hxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes ( GRA4 , GRA6 , ROP7 , and tgd057 ) that encode characterized CD8 + T cell epitopes that elicit corresponding antigen-specific CD8 + T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8 + T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δ gra4 and Δ gra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δ ku80 Δ hxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.