ABSTRACT In low- and middle-income countries cryptosporidiosis is one of the leading causes of debilitating infant diarrhea. Effective drug option to control Cryptosporidium parasites remain limited and efforts to develop a vaccine are at an early stage. In spite of the public health impact of childhood diarrhea, the lack of research tools continues to hamper progress towards a better understanding of these parasites. The goal of this project is to develop a forward genetics method based on random mutagenesis to facilitate basic and applied research on Cryptosporidium parvum, a zoonotic species responsible for about half of human cases of cryptosporidiosis. The term Forward Genetics refers to methods used to identify genetic determinants of specific phenotypes. This approach has expanded our understanding of model organisms, including the taxonomically related species Toxoplasma gondii. The proposed project will impact primarily four areas of Cryptosporidium research. 1) It will advance drug development efforts by identifying genetic loci targeted by anti-Cryptosporidium compounds. 2) It will enable the dissection of the genetic basis of clinically relevant traits such as drug susceptibility, virulence or oocyst output, and potentially uncover genes associated with multidrug resistance. 3) It will contribute to the annotation of genes encoding proteins of unknown function, which still represent a third of the Cryptosporidium genome. 4) The identification of drug resistance mechanisms will facilitate the development of new selectable markers for advancing genome editing procedures. To reduce the frequency of unselected mutations generated by random mutagenesis, and maximize the probability of obtaining selectable C. parvum mutants, two alkylating mutagens and UV irradiation will be used. Mutagenized parasite lines will be selected by backcrossing to wildtype C. parvum in mice while applying drug selection. The genome of multiple drug-resistant C. parvum lines will be sequenced and compared to the genome of the wildtype line to identify mutations under selection. Specific Aim 1 will investigate chemical mutagenesis to generate selectable C. parvum phenotypes. We will select for resistance to two drugs: aurintricarboxylic acid, and the piperazine-based compound MMV665917 from the Medicines for Malaria Venture collection, found to be effective against C. parvum. In Specific Aim 2, UV irradiation will be used to mutagenize oocysts. For both aims, backcrossing to wildtype C. parvum will be used to flush out irrelevant mutations. The genome of multiple, independently selected drug-resistant lines will be sequenced. Mutations correlating with drug resistance in replicated selection experiments will be identified by mapping sequence reads to the C. parvum reference genome. To demonstrate causality, a mutation hypothesized based on replicated selection experiments to confer drug resistance will be introduced into wildtype C. parvum using CRISP/Cas9. A suc...