Abstract. Human cytomegalovirus (HCMV) is the most common cause of congenital viral infections. HCMV’s ability to infect the placenta plays a central role in its pathogenesis during pregnancy. Placental infection can be sufficient to cause adverse pregnancy outcomes and is likely a prerequisite to congenital cytomegalovirus infection. The placenta is resistant to viral infection in part due to the antiviral activity of trophoblasts. These fetal-derived cells form the physical barrier that separates maternal and fetal circulation and secrete a variety of factors, including type III interferon, exosomes, and antimicrobial peptides, that collectively defend the maternal-fetal interface from infection. However, HCMV can replicate in trophoblasts and may injure the placenta either by directly infecting and killing trophoblasts or by stimulating an injurious maternal or fetal immune response. Two preliminary studies have led us to hypothesize that trophoblast differentiation sensitizes the placenta to infection-associated injury late in gestation. Firstly, we have found that human trophoblast stem cells (TSCs) can be infected by HCMV but are not permissive to replication. Transcriptome profiling revealed that, like other embryonic and multipotent stem cells, TSCs constitutively express a subset of interferon stimulated genes (ISGs). Suspecting that one or more of these factors protect TSCs and their derivatives from HCMV during early differentiation, we will conduct an unbiased CRISPR/Cas9 screen to identify HCMV restriction factors in these cells. Follow up studies will use targeted mutagenesis to study how ISG deletion affects the sensitivity of TSCs, TSC-derived trophoblasts, and trophoblast organoids to infection. Separately, studies in a guinea pig model of congenital cytomegalovirus infection revealed that maternal infection after mid-gestation causes a unique pattern of viral infection in the placenta and a transcriptional response that implicates placental immunop