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

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.