Developing a Cre & DiCre conditional gene expression system to investigate essential Cryptosporidium genes

The protozoan parasite, Cryptosporidium, is the causative agent of cryptosporidiosis. It is one of the leading causes of mortality caused by diarrhoeal disease in young children and immunocompromised individuals. Currently, there are no effective treatments or vaccines available. I aim to adapt a regulatable gene expression system to validate potential Cryptosporidium drug targets and explore parasite transmission biology. Specifically, I aim to adapt the next generation of Cre and DiCre conditional gene expression system in Cryptosporidium parvum. I used CRISPR/Cas9 to generate transgenic C. parvum strains that express both NanoLuciferase (NLuc) and Firefly Luciferase (FLuc). These reporter proteins allow tracking of oocyst shedding (NLuc) and parasite infection in the gut (FLuc). CpCDPK1 (Calcium-dependent protein kinase 1) is a well characterised protein and is essential for C. parvum survival. Therefore, I employed it as a positive control for developing my Cre/DiCre system. Using CRISPR/Cas9, loxP sites are introduced into the genome of C. parvum flanking regions of interest, for example the calcium binding domain (CaBD) or the promoter of CpCDPK1. I replaced the promoter of CDPK1 with a promoter that has a similar gene expression profile throughout the C. paruvm life cycle. The Cre/DiCre cassette is introduced at a second locus, and when expressed will excise the region between the loxP sites. We utilise our new selection marker (KRSR) to generate both floxed and Cre/DiCre strains and then select for both mutations by genetic cross. I successfully generated the strains containgin Cre/DiCre machinery and a floxed CpCDPK1 promoter strain. On-going work aims to propagate and validate these strains. Future work will cross these strains to produce a recombinant progeny that can be used to validate CpCDPK1 as an essential gene in Cryptosporidium. This is a framework for evaluating other candidate genes whose essentiality is unknown. For transmission related genes, the comparison of NLuc vs FLuc will aid in determining its role in transmission vs parasite survival in the gut.