CRISPR/Cas9-Mediated Mutagenesis of in

The homeotic gene () 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 in was undertaken using RT-qPCR and the CRISPR/Cas9 genome-editing system. Phylogenetic analyses indicate that shares a high degree of sequence homology among Lepidoptera species. The expression profile of was detected by RT-qPCR. The results showed that was expressed in the whole growth cycle of , 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 was most abundantly expressed in the thoracic segments and legs. To functionally disrupt , two sgRNA sites were designed at the first exon of and the gene was knocked out by CRISPR/Cas9 via microinjection. The results showed that the deletion of 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 resulted in high embryo mortality. Through DNA sequencing, it was found that the target site of the mutant had different degrees of frameshift mutations, indicating that the mutant phenotype was indeed caused by the knockout of .