CRISPR/Cas9-mediated genome editing ofFrankliniella occidentalis,the western flower thrips, via embryonic microinjection

The western flower thrips, Frankliniella occidentalis , poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye-color genes, white ( Fo-w ) and cinnabar ( Fo-cn ), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo-w or Fo-cn specific guide RNAs. Homozygous Fo-w and Fo-cn knockout lines were established by crossing mutant females and males. The Fo-w knockout line revealed an age-dependent modification of eye-color phenotype. Specifically, while young larvae exhibit ivory-colored eyes, the color transitions to bright red as they age. Unexpectedly, loss of Fo-w function also altered body color, with Fo-w mutants having a lighter colored body than wild type, suggesting a dual role for Fo-w in thrips. In contrast, individuals from the Fo-cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene functions and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.