Towards CRISPR/Cas9 Genome Editing in Spodoptera frugiperda : A Proof‐of‐Concept Targeting Wing and Eye Color Genes

Fall armyworm (Spodoptera frugiperda), a globally significant destructive lepidopteran invasive pest, has recently invaded Africa and Asia, threatening food security. Conventional method of management, including chemical insecticides, are often ineffective due to various reasons compelling the need to explore alternative strategies. In this regard, CRISPR/Cas9 based genome editing has emerged as a powerful tool for functional genomics in insects, enabling to introduce site-specific mutations for various purposes. In this study, we applied multiplex CRISPR/Cas9 ribonucleoprotein (RNP) injections to disrupt two key genes in S. frugiperda: the spalt (Sfspalt), which regulates wing patterning, and tryptophan 2,3-dioxygenase (Sfto/vermillion) gene, involved in eye pigmentation. Microinjection of sgRNA/Cas9 ribonucleoprotein complex into freshly laid eggs resulted in distinct phenotypic alterations, including altered wing pigmentation and modified eyespot patterns, as well as golden-yellow eye color mutants. Genotyping and ICE analysis confirmed the presence of frameshift mutations in the target loci, supporting the phenotypic changes. Notably, while mutations were detected, only a single individual was confirmed to carry mutations in both genes simultaneously. These results demonstrate the technical feasibility of multiplex CRISPR/Cas9 editing in S. frugiperda, but also reveal a low frequency of confirmed events under the present experimental conditions. Therefore, this study is considered a proof of concept establishing a preliminary workflow in multiplex platform. The findings provide foundational insights for further optimization of genome editing strategies targeting sex related genes in this agriculturally important pest.