CRISPR/Cas9 Mediated Editing of Bdtektin1 Gene Induces Sterility in Male Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)

Abstract

The Oriental fruit fly, Bactrocera dorsalis (B. dorsalis) is a highly invasive, widely distributed notorious pest restricting global fruit trade immensely. There are several approaches to managing this pest, still require newer approaches. In this regard, recently a novel approach called precision-guided sterile insect technique (pgSIT) is gaining momentum in inducing both female sex elimination or sex conversion and male sterility at one go. Developing a species-specific pgSIT system requires validation of targets such as sex determination and spermatogenesis genes. In this regard, B. dorsalis is highly amenable for area-wide pest management and in the present study, we have validated the loss-of-function of the spermatogenesis-related gene, tektin1 using the CRISPR/Cas9 ribonucleoprotein (RNP) complex. This gene was cloned from the local isolate of B. dorsalis and two promising single guide RNAs (sgRNAs) were designed and validated through in vitro restriction analysis. Injection of the RNP complex (sgRNA + Cas9 protein) into the G0 embryo resulted in three adult males carrying mutations at the target site. The phenotype of the mutants was determined through crossing studies, namely, △1♂ × WT ♀, △2♂ × WT ♀, △3♂ × WT ♀, and WT ♂ × WT ♀ and that showed hatching rates of 0%, 11.70%, 0%, and 45.12%, respectively. The mutant males had more nonviable sperm as compared to control. This study underscores the pivotal role of the Bdtektin1 gene for male fertility and is a promising candidate for further development of pgSIT system for B. dorsalis.