Project summary Diarrheal disease kills 2,195 children each day and persists as the fifth leading cause of death among children under the age of 5, with an especially high burden on low-income countries. Among pathogens, the parasite Cryptosporidium remains a leading cause of diarrhea worldwide and infects millions of people each year. It is the second leading cause of diarrheal disease in infants and is the leading cause of waterborne illness in the United States. Currently, nitazoxanide is the only drug available to treat this parasitic disease, but it is ineffective in curing the most vulnerable populations, including malnourished children and immunocompromised patients. With a burden 2-5 times greater than previously thought, cryptosporidiosis is severely understudied and novel therapeutics are needed to squander this emerging global health threat. Transmission of the parasite occurs via the fecal-oral route, with ingestion of as little as 10 Cryptosporidium oocysts leading to infection. The parasite then progresses through asexual growth, replication, and division in intestinal epithelial cells, followed by transition to a male or female form. Sexual reproduction of male and female parasites results in the production of more infectious oocysts that are shed by the mammalian host. While a few molecular markers have been identified to demarcate this life cycle progression, there is a general lack of knowledge about the signaling pathways and gene expression changes involved in Cryptosporidium development. In related parasites that cause malaria and toxoplasmosis, DNA- binding transcription factors called AP2s drive cell cycle transitions, including sexual commitment, host cell invasion, chronic infection, and deployment of virulence proteins. Preliminary findings suggest that a number of AP2s are differentially expressed between the asexual and sexual stages of C. parvum, although a more thorough genetic analysis and classification is necessary. I hypothesize that AP2 transcription factors drive cell fate decisions at key points during the Cryptosporidium life cycle, such as asexual division and sexual commitment. To investigate this further, I aim to 1) identify stage-specific transcription factors involved in C. parvum life cycle progression and 2) determine their functional roles during development. Using high- throughput genomic technologies, I will examine the gene expression of transcriptional regulators across the cell cycle and prioritize for regulators with distinct expression patterns. I will elucidate their roles in parasite development and differentiation by utilizing CRISPR/Cas9 tools developed in our laboratory to genetically modify C. parvum. Transgenic parasites will be used to study protein expression and localization of the candidate gene as well as the resultant phenotype in conditional knockout experiments. The ability to disrupt these critical regulators of the life cycle will greatly accelerate the development of effect...