Targeted CRISPR Screens Reveal Genes Essential for Cryptosporidium Survival in the Host Intestine

The Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited eKicacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo. Using this method we examine the parasites pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group we determined the parasite gene known as Cp23 to be essential for survival, which was confirmed through inducible knockout in vitro and in vivo. Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is essential for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics.