Osteocalcin (Ocn), encoded in humans by the bone gamma-carboxyglutamic acid-containing protein (BGLAP) gene, is a small protein almost exclusively expressed by mature osteoblasts and osteocytes. The mouse locus expressing osteocalcin consists of a gene cluster where the gene has undergone a triplication resulting in two bone-specific isoforms (Og1 and Og2) and one isoform (Org) not expressed in bone proposed to function in the kidney. Previously, mice homozygous for a genomic deletion encompassing both Og1 and Og2 were created and displayed high bone mass and developed systemic metabolic phenotypes including decreased glucose tolerance and insulin resistance as well as alterations in male fertility and fetal neural development. A caveat to this work is that te completion of the mouse genome subsequent to the generation of these mice revealed the presence of a putative gene within this region (GM6821) that encodes a potentially spliced transcript with homology to members of the progestin and adipoQ receptor family. GM6821 is also removed via the genomic deletion that removes mouse Og1 and Og2. While the Ocn-deficient mice have allowed powerful insights into the role(s) of osteocalcin in mouse physiology, the fact the genomic locus is organized very differently from the human does create potential complications in translating this knowledge to the clinic. While the mouse locus contains a complex triplication, the rat genome is structured similarly to the human locus, carrying a single gene expressing OCN/BGLAP. Thus, genomic alteration of the rat gene would be more analogous to the human situation and the generation of Ocn-deficient rats would provide a powerful means to confirm the findings seen in the mouse models and ensure that the concomitant deletion of GM6821 in the Ocn-deficient mouse is not contributing to the observed phenotypes since the rat and human genomes lack GM6821. A proposal to create Ocn-deficient rats would not have been feasible even a year ago. However, the availability of the CRISPR/Cas9 system completely changes the paradigm in terms of the cost and time required to generate genetically engineered rat models. Specific Aim 1. Use CRISPR/Cas9 technology to create osteocalcin-deficient rats. Our laboratory has used CRISPR/Cas9 to generate mice carrying high frequency biallelic deletions in the murine tyrosinase locus. We will carry out an analogous experiment to target the rat Ocn/BGLAP locus to inactivate the gene. Specific Aim 2. Characterize skeletal and metabolic phenotypes in osteocalcin-deficient rats. After generating Osteocalcin-deficient rats, we will determine if they display skeletal and metabolic phenotypes similar to those observed in mice carrying the large deletion of the osteocalcin locus.