Functional Genomics via Zebrafish CRISPR Reveals Pathogenic Landscape of 16 Candidate Genes in Hemifacial Microsomia

Functional Genomics via Zebrafish CRISPR Reveals Pathogenic Landscape of 16 Candidate Genes in Hemifacial Microsomia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Functional Genomics via Zebrafish CRISPR Reveals Pathogenic Landscape of 16 Candidate Genes in Hemifacial Microsomia Zhifeng Li, Zhang Zhiyong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7317799/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Hemifacial Microsomia (HFM) is a genetically complex craniofacial disorder. While GWAS and family studies have identified multiple candidate genes, functional validation rates remain low (<10%). Methods: We established a high-throughput zebrafish CRISPR-Cas9 platform to functionally validate 16 prioritized genes (12 literature-derived, 4 bioinformatically predicted). Tg(col2a1a:EGFP) embryos underwent F0 knockout with ≥70% editing efficiency. Mandibular development was quantitatively analyzed using six morphometric parameters at 5 dpf. Results: We identified three high-confidence pathogenic genes: EDNRB knockout caused pan-mandibular hypoplasia (Meckel's cartilage ↓21%, p<0.0001); FGF3 deficiency led to selective arch defects (ceratohyal length ↓28%, p<0.0001); EPAS1 ablation resulted in unilateral dysgenesis (cranial length ↓24%, p<0.0001). PAX1 knockout induced lethal pan-craniofacial defects. Conclusion: This study establishes EDNRB-FGF3-EPAS1 as a core pathogenic axis and validates environmental susceptibility genes (TP53/ESR2). These findings enable: 1) OMENS+ molecular subtyping, 2) gene-targeted therapeutic strategies, and 3) prenatal risk assessment for environmental exposures. Biological sciences/Developmental biology Health sciences/Diseases Biological sciences/Genetics Health sciences/Medical research Biological sciences/Molecular biology Hemifacial Microsomia Craniofacial Development Zebrafish CRISPR Screening Mandibular Morphometrics Genetic Pathogenesis Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7317799","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":518780120,"identity":"83468cac-8a90-41df-9ee8-6ed023053d97","order_by":0,"name":"Zhifeng Li","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhifeng","middleName":"","lastName":"Li","suffix":""},{"id":518780121,"identity":"6db17b32-f2b7-443c-bdc2-14924ee1ed82","order_by":1,"name":"Zhang 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While GWAS and family studies have identified multiple candidate genes, functional validation rates remain low (\u0026lt;10%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethods: We established a high-throughput zebrafish CRISPR-Cas9 platform to functionally validate 16 prioritized genes (12 literature-derived, 4 bioinformatically predicted). Tg(col2a1a:EGFP) embryos underwent F0 knockout with ≥70% editing efficiency. Mandibular development was quantitatively analyzed using six morphometric parameters at 5 dpf.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Results: We identified three high-confidence pathogenic genes: EDNRB knockout caused pan-mandibular hypoplasia (Meckel's cartilage ↓21%, p\u0026lt;0.0001); FGF3 deficiency led to selective arch defects (ceratohyal length ↓28%, p\u0026lt;0.0001); EPAS1 ablation resulted in unilateral dysgenesis (cranial length ↓24%, p\u0026lt;0.0001). 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