Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility

• DOI: 10.7554/elife.100060.3
• Publication Date: 2025-01-16T10:15:45Z
• Authors: Sophie Debaenst; Tamara Jarayseh; Hanna De Saffel; Jan Willem Bek; Matthieu Boone; Ivan Josipovic; Pierre Kibleur; Ronald Y Kwon; Paul J Coucke; Andy Willaert
• Keywords: Danio rerio; GENETICS; QH301-705.5; Science; Q; R; Biology and Life Sciences; Genetics and Genomics; OSTEOPOROSIS; GENOME; Medicine and Health Sciences; Medicine; genetics; Biology (General); Crispants; Zebrafish

Heritable fragile bone disorders (FBDs), ranging from multifactorial to rare monogenic conditions, are characterized by an elevated fracture risk. Validating causative genes and understanding their mechanisms remain challenging. We assessed a semi-high throughput zebrafish screening platform for rapid in vivo functional testing of candidate FBD genes. Six linked severe recessive osteogenesis imperfecta (OI) four associated with mineral density (BMD) genome-wide association studies were analyzed using CRISPR/Cas9-based crispant F0 mosaic founder zebrafish. Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, 90 days post-fertilization (dpf) microscopy, Alizarin Red S staining, microCT was performed. Larval crispants showed variable osteoblast mineralization phenotypes, while adult displayed consistent skeletal defects, including malformed neural haemal arches, vertebral fractures fusions, altered volume density. In addition, aldh7a1 mbtps2 experienced increased mortality due deformities. RT-qPCR revealed differential expression osteogenic markers bglap col1a1a , highlighting biomarker potential. Our results establish as robust tool gene validation, combining molecular analyses across developmental stages uncover novel insights into functions biology.