Utilizing CRISPR-Cas13d-knockdown in zebrafish to study a rare monogenic bone fragility syndrome.

Pathogenic variants in the human gene coding for SMS2 protein result in a rare form of primary osteoporosis, "Calvarial doughnut lesions with bone fragility" (CDL). To obtain a better understanding of the gene's function and the molecular mechanisms underlying CDL, we studied mRNA expression of the zebrafish orthologs for , and , in WT zebrafish and developed zebrafish models using the novel CRISPR-Cas13d-knockdown. The , , and knockdown zebrafish were analyzed by embryo phenotype at 2 d post-fertilization and cartilage and bone staining at 7 d post-fertilization. In situ hybridization studies of embryonic and early larval WT zebrafish demonstrated expression in the brain, myotome, and craniofacial skeletal and cartilage elements, and expression in the myotome and craniofacial cartilage elements. Single-cell RNA sequencing of juvenile WT zebrafish calvaria cells detected high expression in osteogenic cells. Knockdown of and in zebrafish had detrimental effect on embryonic development and compromised notochord and craniofacial formation. Our findings provide novel information on the role of in the musculoskeletal system. The CRISPR-Cas13d-knockdown zebrafish models for CDL serve as preliminary platforms for exploring embryonic and early larval gene function and obtaining clues for molecular mechanisms underlying its pathology.KEYWORDScalvarial doughnut lesions with bone fragility, early-onset osteoporosis, sphingomyelin metabolism, sphingomyelin synthase 2, zebrafish© The Author(s) 2025. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.