C‐Terminal End of Gli3 Is Critical for Functional Protein Synthesis and Gli3‐Dependent Anatomical Development

The GLI3 gene, a pivotal component of the hedgehog (HH) signaling pathway, plays a fundamental role in the development and patterning of various body structures, including the brain and limbs. Mutations in the GLI3 gene, particularly in the C‐terminal domain, are implicated in congenital anomalies such as Greig cephalopolysyndactyly syndrome and Pallister–Hall syndrome. Recent studies have also suggested an archaic human‐type mutation in the C‐terminal end, which altered downstream gene regulations and anatomical structures in mice. However, the biological effects of the disruption in the Gli3 C‐terminal end have not been studied well. Here we report novel Gli3 mutant mice with nonsense mutations in the C‐terminal end using CRISPR/Cas12a‐mediated genome editing. Analysis of the genotype–phenotype correlations has revealed that the C‐terminal end of Gli3 is critical for functional protein synthesis; therefore, the disruption of this region causes severe abnormalities in brain and digit formation. These results provide insight into the mechanisms by which GLI3 mutants can cause adverse consequences during human development or result in diverse phenotypes during evolution.