Toxoplasma gondii is a model apicomplexan that also causes severe disease in fetuses and immuno- compromised individuals. Many Toxoplasma genes likely evolved to modulate the host immune system, overcome in vivo nutrient deficiencies, and disseminate to distant organs. The key to Toxoplasma’s successful co-option of the host are proteins secreted from its rhoptry and dense granule secretory organelles, named ROPs and GRAs, respectively. We, therefore, hypothesize that a systematic analysis of Toxoplasma genes that contribute to in vivo fitness will help the identification of critical genes for Toxoplasma pathogenesis and also provide new insights into its dissemination. As a proof of concept, we have performed CRISPR/Cas9 loss- of-function screens in the type I RH strain to determine the contribution of Toxoplasma putative GRA-encoding genes to fitness in mice. This study identified Toxoplasma genes that are important for fitness at the site of infection or required to reach organs. In this proposal we will build on these preliminary data to optimize in vivo loss-of-function screens in the virulent type I RH and the cyst-forming type II ME49 strains. In aim 1 we will optimize ROP- and GRA-gene targeting libraries for in vivo loss-of-function screens in the widely used virulent type I RH and cyst-forming type II ME49 strains. We will do this by optimizing the number of genes to be targeted and the number of single guide (sg)RNAs targeting each gene. We will use these sgRNA libraries to make pools of loss-of-function mutants in both virulent type I RH and cyst-forming type II ME49 strains. We will i.p. infect mice with these mutants and determine the fitness of each mutant at the site of infection and in organs. In aim 2 we will confirm 5 Toxoplasma genes that are important for in vivo fitness at the site of infection and 5 genes important for dissemination to distant organs. We will determine the localization of the gene product in Toxoplasma, generate knockout and complemented parasites and confirm the in vivo fitness defect. This proposal will advance the field by: 1) providing optimized sgRNA libraries and optimized methodology for performing in vivo loss-of-function screens in different parasite genetic backgrounds that are widely used by the Toxoplasma community; 2) identifying novel Toxoplasma genes important for in vivo fitness at the site of infection and dissemination to distant organs.