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Parasite invasion and egress cause damage to intestinal epithelial cells, which is mediated by a variety of secretory proteins from several unique organelles, such as micronemes. Previous spatial proteomic analysis has identified insulinase-like protease 6 (INS6) as a putative microneme protein in <jats:italic>C. parvum<\/jats:italic>. However, the functional contribution of INS6 to <jats:italic>Cryptosporidium<\/jats:italic> pathogenicity remains poorly characterized. In this study, we used genetic manipulation techniques to investigate the expression and biological functions of INS6 in <jats:italic>C. parvum<\/jats:italic>.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methodology\/principal findings<\/jats:title>\n<jats:p>The <jats:italic>INS6<\/jats:italic> gene was tagged and deleted in <jats:italic>C. parvum<\/jats:italic> using CRISPR\/Cas9 technology. The expression of <jats:italic>INS6<\/jats:italic> was determined by immunofluorescence analysis, ultrastructure-expansion microscopy, and immunoelectron microscopy. Endogenous labelling showed low levels of INS6 expression, which is found in <jats:italic>C. parvum<\/jats:italic> micronemes and is absent during the male gamont stage. The effect of <jats:italic>INS6<\/jats:italic> deletion on parasite growth and pathogenicity was assessed <jats:italic>in vitro<\/jats:italic> using HCT-8 cultures and <jats:italic>in vivo<\/jats:italic> by infection of interferon-\u03b3 knockout mice. Deletion of the <jats:italic>INS6<\/jats:italic> gene impaired <jats:italic>C. parvum<\/jats:italic> proliferation <jats:italic>in vitro<\/jats:italic> and significantly reduced the parasite burden in infected mice. In addition, mice infected with the <jats:italic>\u0394ins6<\/jats:italic> strain showed a significant reduction in the intestinal villus-to-crypt ratio, attenuated body weight loss and increased survival rates, compared to those infected with the INS6\u20133HA strain.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Conclusions\/significance<\/jats:title>\n<jats:p>These results indicate that INS6 protein is involved in <jats:italic>C. parvum<\/jats:italic> proliferation and plays a critical role in modulating the pathogenicity of this zoonotic parasite. Deletion of this gene affects the invasion efficiency and pathogenicity of the parasite.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0013532","type":"journal-article","created":{"date-parts":[[2025,9,12]],"date-time":"2025-09-12T17:56:22Z","timestamp":1757699782000},"page":"e0013532","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Cryptosporidium parvum protease INS6 plays an important role in parasite proliferation and 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