Knockout of the INS 19 or INS 20 gene in the IIdA20G1 subtype of Cryptosporidium parvum

This study aims to investigate the expression and biological functions of insulinase like proteins INS 19 and INS 20 in Cryptosporidium parvum. Using CRISPR Cas9 technology, we separately knocked out the INS 19 and INS 20 genes to generate corresponding mutants. Immunofluorescence analysis revealed that INS 19 and INS 20 are expressed at different developmental stages of the pathogen. Although knocking out these genes did not significantly affect in vitro growth, INS 20 gene knockout, deletion of multiple functional domains, or mutation of active motifs significantly reduced C. parvum infection intensity in gamma interferon knockout mice, leading to reduced intestinal damage and parasite burden. These findings suggest that INS 19 and INS 20 are expressed at specific developmental stages and have distinct biological functions, with INS 20 playing a critical role in the zoonotic infection and adaptation of C. parvum.