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The ESCRT machinery (Endosomal Sorting Complex Required for Transport), consists of four multi-subunit complexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, that play a role in the transport of ubiquitinated cargoes to intraluminal vesicles that bud from multivesicular bodies derived of endosomal compartments. These multivesicular bodies later fuse with lysosomes for degradation of the endocytosed cargoes or with the plasma membrane secreting intraluminal vesicles as a heterogeneous mixture of exosomes. Because Trypanosomatids, which are protozoan parasites that secrete extracellular vesicles and depends on endocytosis, here we analyzed the role of ESCRT complex in Trypanosoma cruzi, the agent of Chagas disease. Parasites expressing the Cas9 and T7 RNA polymerase were transfected with DNA fragments coding for two sgRNA that pair the VPS23 gene, a member of the ESCRT-I, together with DNA donor fragments to replace the entire gene by hygromycin and blasticidin resistance markers. Only one of the VPS23 alleles were replaced by one copy of the resistance markers, suggesting that VPS23 is essential for T. cruzi. Similar results were obtained for Leishmania mexicana. These partial knockouts significantly lower the receptor mediated endocytosis of transferrin, but not the fluid phase BSA-uptake in T. cruzi. In addition, they reduced the secretion of extracellular vesicles by epimastigotes. The partial knockout did not affect epimastigote proliferation and metacyclogenesis, and the resulting trypomastigotes were able to infect cells as the parental cell lines. We are currently analyzing whether the secretion of extracellular vesicles is also changed in trypomastigotes released from infected mammalian cells and the capacity of these parasites to promote infection. These results demonstrated the essential role of TcVPS23 by modulating endocytic and secretory activities in T. cruzi, and might be useful to understand the role of extracellular vesicles, know to play an important role in the parasite virulence. [ABSTRACT FROM AUTHOR]