Summary: Chagas disease, an infectious disease caused by the protozoan parasite Trypanosoma cruzi, affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are urgently needed to combat this disease. Phosphorylation-mediated signal transduction in eukaryotes plays key roles as regulator of protein function. Considering the changes in phosphorylation patterns reported in T. cruzi developmental stages, it is likely that protein kinases play a pivotal role in the regulation of cell cycle, metabolism, survival, and particularly in parasite differentiation. Therefore, protein kinases, which represent near 2% of the T. cruzi genome, conform an attractive group of molecular targets for antiparasitic interventions. In addition, the subcellular localization of protein kinases is determinant for their activity and function. Therefore, elucidating their localization is crucial to predict their participation in specific cellular processes. Programmable CRISPR/Cas9 systems have improved our ability to produce precise genome manipulations in T. cruzi. However, current methodologies have limitations to further perform the simultaneous analysis of multiple knockout mutants in T. cruzi, which also restricts the identification of molecular targets for drug discovery. Here we will apply a protein kinase screening by gene knockout and gene tagging in T. cruzi epimastigotes, to identify protein kinases involved in life cycle progression. We will investigate 124 protein kinases of T. cruzi, most of them with no apparent orthologs in mammals. We will analyze the phenotype of pooled knockout mutants harboring unique DNA barcodes that will be generated by CRISPR/Cas9-based methodology. Furthermore, we will use next-generation sequencing to identify protein kinases required for epimastigote survival, growth and differentiation into metacyclic trypomastigotes, and for T. cruzi infection and proliferation in tissue-cultured host cells. Overall, we will perform a large-scale screening to assess parasite fitness. We will also perform endogenous tagging of selected genes encoding protein kinases to determine their subcellular localization. Elucidating the role of protein kinases in T. cruzi will allow to gain insight organelle-associated signaling pathways required for parasite survival, proliferation and differentiation.