Variants in NOD2 are the strongest genetic link to the development of Crohn’s disease (CD) and evidence suggests that NOD2 activation by its ligand, muramyl dipeptide (MDP), a component of bacterial peptidoglycan (PGN), mounts a protective response at the level of the intestinal epithelium. In line with this, a recent study showed low abundance of a gene family of PGN-hydrolases, which participate in cleaving PGN to form MDP, in the microbiome of CD patients and correspondingly, diminished fecal levels of MDP. However, whether these changes were a cause or consequence of disease is unknown. Our team has a unique opportunity to investigate this question from a potential causal perspective. Leveraging metagenomic data and human stool samples from the Crohn’s and Colitis Canada Genetic, Environmental, Microbial project (GEM; gemproject.ca), a prospective study to determine future risk of CD in healthy first-degree relatives (FDRs) of CD patients, we hypothesize that low gene abundance of specific PGN-hydrolases correlates with diminished NOD2-ligand bioavailability in the microbiota and can predict the development of CD. Our approach will define PGN-hydrolase gene abundance and NOD2-ligand bioavailability as pre-disease biomarkers. Moreover, modulation of NOD2-ligand bioavailability will be explored as a protective therapeutic option for the treatment of CD.