A Novel Method to Silence Genes in Cryptosporidium

A major bottleneck for developing new drugs and vaccines against cryptosporidiosis has been the lack of methods to study gene function in this parasite. Silencing of genes using small interference RNA (siRNA) is a powerful methodology to investigate gene function in multiple organisms and has been widely used to identify drug targets for multiple pathogens. Cryptosporidium does not possess the enzymes of the classical siRNA pathway. Therefore, its genes cannot be silenced by standard siRNA technology. To circumvent that problem, we developed a strategy to knock down Cryptosporidium mRNA by reconstituting the effector arm of the siRNA pathway using recombinant enzymes and synthetic single-strand RNA (ssRNA). We have induced silencing of several genes in Cryptosporidium by transfecting parasites with hybrid complexes formed between recombinant human Argonaute (hAgo2) and Cryptosporidium ssRNA. We have used this methodology to study the role of selected genes in host-parasite interactions. It also can be used to identify potential targets for chemotherapy. Standardized protocols to silence genes in Cryptosporidium parasites are described in this chapter. In addition, we described the use of ssRNA/Ago complexes as a novel strategy to reduce Cryptosporidium infection.