Giardia lamblia is a widespread protozoan parasite of humans and animals causing significant diarrheal disease worldwide. Giardia cysts are transmitted between hosts through feces. Once ingested, cysts transform into trophozoites that colonize the duodenal epithelium – a region of varied nutrients, redox stress, and immune responses. Unknown external cues in the gut trigger trophozoites to encyst, and then cysts are passed to new hosts. Giardia is also reported to have a quasi-meiotic stage, termed diplomixis, that occurs in late encystation and is characterized by fusing and exchange of DNA between cyst nuclei. Due to a historical lack of molecular genetic tools, the pathogenesis and basic biology of Giardia is grossly understudied. Developmental transitions and environmental responses are commonly mediated by transcription factors (TFs) – modular proteins that often contain DNA binding domains (DBDs). However, only 36 TFs with DBDs are predicted in the Giardia genome as compared to about 200 predicted in similar sized eukaryotic genomes. Over 500 proteins have more general motifs such as basic leucine-zipper domains, and these can also function as TFs. Only a handful of Giardia TFs have been studied in any detail; all are associated with early to mid-encystation. Basal TFs, redox responsive TFs, late encystation/diplomixis TFs, or cell cycle related TFs have not yet been identified. Our lack of knowledge of the identities and functions of Giardia TFs has severely limited our understanding of genetic regulatory networks throughout the Giardia life cycle, particularly those associated with cell division and pathogenesis. To address these deficits, we will use an unbiased, high-throughput, genome-wide yeast one-hybrid (Y1H) screen to identify additional DNA binding proteins likely representing candidate TFs (Aim 1). In many systems, such screens are commonly used to define genome-wide protein-DNA interactions. Using robotic screening of arrayed “prey” libraries, we will screen a total of nine promoter “baits” (three constitutive loci; three redox stress- associated loci, and three encystation loci) against three “prey” cDNA libraries (constitutive; redox stress; late encystation/diplomixis) for a total of 27 (3 x 3 x 3) combinations. Both predicted DBD proteins and candidate TFs identified in the Y1H screen will be additionally prioritized by CRISPRi mediated knockdown, with phenotypic screening for defects in growth, mid-encystation and genetic marker exchange in cysts (diplomixis), and redox stress. Knockdowns of candidate TFs with strong defects will be further characterized using promoter-luciferase assays and a novel dCas-TF luciferase fusion assay to evaluate transcriptional activation or repression (Aim 2). Overall, this project will define and prioritize Giardia TFs as groundwork for future interrogation of Giardia regulatory networks throughout the life cycle.