| Summary: | The homeotic gene <i>Antennapedia</i> (<i>Antp</i>) has been identified as playing a pivotal role in the morphogenesis of the thorax and wings across various insect species. Leveraging insights from previous studies, the functional characterization of <i>Antp</i> in <i>S. frugiperda</i> was undertaken using RT-qPCR and the CRISPR/Cas9 genome-editing system. Phylogenetic analyses indicate that <i>Antp</i> shares a high degree of sequence homology among Lepidoptera species. The expression profile of <i>SfAntp</i> was detected by RT-qPCR. The results showed that <i>SfAntp</i> was expressed in the whole growth cycle of <i>S. frugiperda</i>, the expression level was the highest in the egg stage, and the expression level was higher from 12 h to 48 h. Tissue-specific expression profiling demonstrated that <i>SfAntp</i> was most abundantly expressed in the thoracic segments and legs. To functionally disrupt <i>SfAntp</i>, two sgRNA sites were designed at the first exon of <i>SfAntp</i> and the gene was knocked out by CRISPR/Cas9 via microinjection. The results showed that the deletion of <i>SfAntp</i> produced a mutant phenotype of thoracic fusion, thoracic leg defect, leg-like protrusions between the head and thoracic segments and pupation deformity. In addition, deletion of <i>SfAntp</i> resulted in high embryo mortality. Through DNA sequencing, it was found that the target site of the <i>SfAntp</i> mutant had different degrees of frameshift mutations, indicating that the mutant phenotype was indeed caused by the knockout of <i>SfAntp</i>.
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