Amelogenin is the most abundant matrix protein guiding hydroxyapatite formation in enamel, the durable bioceramic tissue that covers vertebrate teeth. Here, we sought to refine structure-function for an amelogenin domain based on in vitro data showing a 42 amino acid amelogenin-derived peptide (ADP7) mimicked formation of hydroxyapatite similar to that observed for the full-length mouse 180 amino acid protein. In mice, we used CRISPR-Cas9 to express only ADP7 by the native amelogenin promoter. Analysis revealed ADP7 messenger RNA expression in developing mouse teeth with the formation of a thin layer of enamel. In vivo, ADP7 peptide partially replaced the function of the full-length amelogenin protein and its several protein isoforms. Protein structure-function relationships identified through in vitro assays can be deployed in whole model animals using CRISPR-Cas9 to validate function of a minimal protein domain to be translated for clinical use as an enamel biomimetic.