Chagas disease, caused by , is a neglected tropical disease with few options for treatment and no available vaccine. Deletion mutants for live attenuated vaccines, particularly deletions of proteins related to the cytoskeleton, have been widely tested in related parasites but candidates have not been tested in . Kharon is one such protein, identified as being associated with the cytoskeleton in and essential for amastigote replication. Here we investigated the Kharon ortholog (Kharon) to test if it has orthologous function and thus potential in generating a live attenuated vaccine. In silico analysis predicted Kharon to be an intrinsically disordered protein, consistent with its ortholog feature, and GFP fusion protein revealed that Kharon is associated with the cytoskeleton of epimastigotes. CRISPR-Cas9-mediated gene disruption impaired epimastigote proliferation and cytokinesis, resulting in altered nucleus-to-kinetoplast ratios and pronounced morphological defects, particularly in the posterior cell region. Despite these abnormalities, Kharon mutants retained the ability to differentiate into metacyclic trypomastigotes and exhibited in vitro infection rates comparable to wild-type parasites. Our data show that Kharon is crucial for cell morphology. However, in contrast to close related parasites, Kharon is not essential for in vitro infectivity.