Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility
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 rap...
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eLife Sciences Publications Ltd
2025-01-01
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| Online Access: | https://elifesciences.org/articles/100060 |
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| author | Sophie Debaenst Tamara Jarayseh Hanna De Saffel Jan Willem Bek Matthieu Boone Ivan Josipovic Pierre Kibleur Ronald Y Kwon Paul J Coucke Andy Willaert |
| author_facet | Sophie Debaenst Tamara Jarayseh Hanna De Saffel Jan Willem Bek Matthieu Boone Ivan Josipovic Pierre Kibleur Ronald Y Kwon Paul J Coucke Andy Willaert |
| author_sort | Sophie Debaenst |
| collection | DOAJ |
| description | 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 genes linked to severe recessive osteogenesis imperfecta (OI) and four associated with bone mineral density (BMD) from genome-wide association studies were analyzed using CRISPR/Cas9-based crispant screening in F0 mosaic founder zebrafish. Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, and 90 days post-fertilization (dpf) using microscopy, Alizarin Red S staining, and microCT was performed. Larval crispants showed variable osteoblast and mineralization phenotypes, while adult crispants displayed consistent skeletal defects, including malformed neural and haemal arches, vertebral fractures and fusions, and altered bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR revealed differential expression of osteogenic markers bglap and col1a1a, highlighting their biomarker potential. Our results establish zebrafish crispant screening as a robust tool for FBD gene validation, combining skeletal and molecular analyses across developmental stages to uncover novel insights into gene functions in bone biology. |
| format | Article |
| id | doaj-art-c7906fcda80340b2bcde0b6c71084bae |
| institution | Kabale University |
| issn | 2050-084X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | eLife Sciences Publications Ltd |
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| series | eLife |
| spelling | doaj-art-c7906fcda80340b2bcde0b6c71084bae2025-01-17T15:06:34ZengeLife Sciences Publications LtdeLife2050-084X2025-01-011310.7554/eLife.100060Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragilitySophie Debaenst0https://orcid.org/0000-0001-7598-919XTamara Jarayseh1Hanna De Saffel2Jan Willem Bek3Matthieu Boone4Ivan Josipovic5Pierre Kibleur6Ronald Y Kwon7https://orcid.org/0000-0001-9760-3761Paul J Coucke8Andy Willaert9https://orcid.org/0000-0002-9543-1932Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, BelgiumCenter for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, BelgiumCenter for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, BelgiumCenter for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, BelgiumCenter for X-ray Tomography, Department of Physics and Astronomy, Ghent University, Ghent, BelgiumCenter for X-ray Tomography, Department of Physics and Astronomy, Ghent University, Ghent, BelgiumCenter for X-ray Tomography, Department of Physics and Astronomy, Ghent University, Ghent, BelgiumDepartment of Orthopaedics and Sports Medicine, University of Washington, Seattle, United States; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, United StatesCenter for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, BelgiumCenter for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, BelgiumHeritable 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 genes linked to severe recessive osteogenesis imperfecta (OI) and four associated with bone mineral density (BMD) from genome-wide association studies were analyzed using CRISPR/Cas9-based crispant screening in F0 mosaic founder zebrafish. Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, and 90 days post-fertilization (dpf) using microscopy, Alizarin Red S staining, and microCT was performed. Larval crispants showed variable osteoblast and mineralization phenotypes, while adult crispants displayed consistent skeletal defects, including malformed neural and haemal arches, vertebral fractures and fusions, and altered bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR revealed differential expression of osteogenic markers bglap and col1a1a, highlighting their biomarker potential. Our results establish zebrafish crispant screening as a robust tool for FBD gene validation, combining skeletal and molecular analyses across developmental stages to uncover novel insights into gene functions in bone biology.https://elifesciences.org/articles/100060geneticsCrispantsDanio rerio |
| spellingShingle | Sophie Debaenst Tamara Jarayseh Hanna De Saffel Jan Willem Bek Matthieu Boone Ivan Josipovic Pierre Kibleur Ronald Y Kwon Paul J Coucke Andy Willaert Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility eLife genetics Crispants Danio rerio |
| title | Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility |
| title_full | Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility |
| title_fullStr | Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility |
| title_full_unstemmed | Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility |
| title_short | Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility |
| title_sort | crispant analysis in zebrafish as a tool for rapid functional screening of disease causing genes for bone fragility |
| topic | genetics Crispants Danio rerio |
| url | https://elifesciences.org/articles/100060 |
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