Functional Genomics Through Zebrafish CRISPR Prioritizes Candidate Genes for Hemifacial Microsomia.

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

The authors 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 6 morphometric parameters at 5 dpf.

RESULTS

The authors identified distinct phenotypic profiles among candidate genes: ednrb knockout caused panmandibular hypoplasia (Meckel 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). In addition, gbx2 and pax1 knockouts showed significant craniofacial anomalies.

CONCLUSION

This study utilizes functional genomics to prioritize EDNRB, FGF3, and EPAS1 as high-confidence candidate genes for hemifacial microsomia. Our findings suggest a functional landscape where distinct genetic pathways converge to produce HFM-like phenotypes and validate TP53 and ESR2 as potential environmental susceptibility factors. These findings enable gene-targeted therapeutic strategies and prenatal risk assessment.