Crohn’s disease (CD) is a specific type of inflammatory bowel disease that occurs in patients of all ages, with the distal region of the small intestine (referred to as ileum) as one of the most commonly affected locations. It afflicts nearly a million people in the United States and is dramatically increasing in prevalence worldwide. Notably, a substantial proportion of CD patients (~25%) are childhood- or adolescentonset, referred to as pediatric CD, which often display highly aggressive symptoms and more complicated clinical course compared to late-onset, adult CD. Large-scale genetic studies of human populations have revealed >200 risk loci in the genome that are associated with CD, the vast majority of which occur in non-coding regions. Although it is widely conjectured that these likely impact regulatory elements, this has not yet been definitively demonstrated for most loci. Moreover, the extent to which the risk loci may affect gut development is unknown and poorly explored, but merits investigation given the importance of developmental aberrations to other inflammatory disorders of the gut. Notably, intractable congenital diarrhea in infants was very recently linked to the deletion of a regulatory element that is transiently active during prenatal gut development. This study aims to (1) define CD-risk loci harboring regulatory elements that are transiently active in pre-natal development and (2) provide functional evidence for a specific CD risk allele affecting regulatory element activity during early gut development, with the ultimate goal of offering a novel perspective on the etiology of pediatric CD. In the first Aim, to study the contribution of CD-associated single nucleotide polymorphisms (SNPs) to early gut development, we will leverage the recent technology of directed differentiation from human pluripotent stem cells (hPSCs) into human intestinal spheroids followed by maturation into organoids (HIOs). This model exhibitsmolecular features closely resembling the primary human fetal gut. To define active regulatory elements genome-wide, we will apply a cutting-edge technology called chromatin run-on sequencing (ChRO-seq) to the hPSC-HIO model as well as to pediatric and adult CD and non-CD ileal biopsies. We will then define those regulatory elements that overlap CD risk loci and are active only in the pre-natal stages of the hPSC-HIO model but not in the post-natal pediatric/adult tissue. In the second Aim, we will define the role of a specific CD-SNP containing regulatory element within the PRDM1 locus during early gut development. In mouse, Prdm1 regulates gut maturation, including the development of Paneth cells, which play important roles in CD pathobiology. We have identified a regulatory element within the human PRDM1 gene that contains a CD-associated SNP and is active only in pre-natal stages and not in adult CD or non-CD ileum. We will use CRISPR/Cas9 single-nucleotide base editing technology to examine the impact of different alleles of this CD-SNP during the 28-day maturation process of ileal HIOs. Successful completion of the proposal will promote child health by providing translational knowledge about the gene regulatory mechanisms of human gut development and pediatric CD pathobiology.