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

Abstract

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 forF. occidentalishampers 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 inF. occidentalisusing the CRISPR/Cas9 genome editing system. Two eye-color genes,white(Fo-w) andcinnabar(Fo-cn), frequently used to assess Cas9 function in insects were identified in theF. occidentalisgenome and targeted for knockout through embryonic microinjection of Cas9 complexed withFo-worFo-cnspecific guide RNAs. HomozygousFo-wandFo-cnknockout lines were established by crossing mutant females and males. TheFo-wknockout 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 ofFo-wfunction also altered body color, withFo-wmutants having a lighter colored body than wild type, suggesting a dual role forFo-win thrips. In contrast, individuals from theFo-cnknockout 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.