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

• DOI: 10.7554/elife.100060.1
• Publication Date: 2024-10-07T16:25:30Z
• Authors: Sophie Debaenst; Tamara Jarayseh; Hanna de Saffel; Jan Willem Bek; Matthieu Boone; Ivan Josipovic; Pierre Kibleur; Ronald Young 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) encompass a spectrum of conditions, from widespread multifactorial to rare monogenic diseases, all characterized by an elevated risk fractures. The process validating causative genes and elucidating their pathogenic mechanisms remains daunting resource-intensive task. In this study, we evaluated the feasibility semi-high throughput zebrafish screening platform for rapid validation in vivo functional testing candidate disease-causing wide range heritable FBDs. Six associated with severe recessive forms Osteogenesis Imperfecta (OI) four BMD, key osteoporosis indicator, identified through genome-wide association studies (GWAS) were selected. crispant approach, based on CRISPR/Cas9 technology, was used phenotype directly F0 mosaic founder zebrafish. Next-Generation Sequencing (NGS) analysis revealed mean indel efficiency 88% across ten different crispants, indicating high proportion knock-out alleles thus resembling stable models. We applied multiple techniques evaluate skeletal characteristics at 7, 14 90 days post-fertilization (dpf), including microscopy osteoblast reporter visualization mineralization Alizarin Red S staining, microCT quantitative analysis. While larval crispants exhibited variable differences osteoblast-positive mineralized surface areas, adult-stage displayed more pronounced consistent phenotypes. Notably, developed malformed neural haemal arches, majority presenting vertebral fractures fusions, some showing significant alterations bone volume density. addition, aldh7a1 mbtps2 experienced increased mortality due deformities. RT-qPCR differentiation formation markers stages indicated differential expression osteogenic bglap col1a1a substantial portion hinting utility as biomarkers FBD screening. summary, our findings demonstrate that offers viable efficient strategy assessment genes. advocate comprehensive approach integrates various evaluates distinct molecular profiles developmental adult stages. This methodology has potential provide new insights into role these biology.