A novel polymerase III promoter for gene editing in the agricultural pest <i>Ceratitis capitata</i>

Abstract We report the identification and functional validation of a 7SK RNA polymerase III promoter in the Mediterranean fruit fly, Ceratitis capitata . CRISPR/Cas9-based genetic control strategies for this global agricultural pest, including gene drives and precision guided sterile insect approaches, require efficient guide RNA expression, yet only a single U6 Pol III promoter had previously been validated for this purpose in C. capitata , and no 7SK promoter had been characterised in any Tephritid species. Using comparative genomics with Drosophila orthologues, we identified a previously unannotated 7SK gene in the C. capitata genome, confirmed its transcriptional activity by RT-PCR, and demonstrated that the cloned promoter drives functional guide RNA expression in CRISPR/Cas9-mediated knockouts of the white gene. Comparative analysis identified putative 7SK orthologues across the Tephritid fruit flies. The availability of this additional new Pol III promoter will enable multiplexed guide RNA strategies using distinct promoters, supporting more robust genetic control designs. Graphical Abstract