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

Pathogenic variants in the human SGMS2 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 SGMS2 gene's function and the molecular mechanisms underlying CDL, we studied mRNA expression of the zebrafish orthologs for SGMS2, sgms2a and sgms2b, in WT zebrafish and developed zebrafish models using the novel CRISPR-Cas13d-knockdown. The sgms2a, sgms2b, and sgms2a+b 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 sgms2a expression in the brain, myotome, and craniofacial skeletal and cartilage elements, and sgms2b expression in the myotome and craniofacial cartilage elements. Single-cell RNA sequencing of juvenile WT zebrafish calvaria cells detected high sgms2a expression in osteogenic cells. Knockdown of sgms2a and sgms2b in zebrafish had detrimental effect on embryonic development and compromised notochord and craniofacial formation. Our findings provide novel information on the role of SGMS2 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.