Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of white gene in melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) produced white eye phenotype.

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNA to target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

CRISPR/Cas9-mediated mutagenesis of Sex lethal (Sxl) gene impacts fertility of the Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

Identification of novel approaches for managing the global pest, the Fall armyworm, Spodoptera frugiperda, is the need of the hour, as it defies many management strategies including synthetic chemicals, Bt transgenics, and so on. Recently CRISPR/Cas9-based genome editing opened up newer avenues to design novel pest management strategies such as precision-guided sterile insect technique (pgSIT). In this regard, genes governing sex determination, egg reproduction, and spermatogenesis could be the prime targets for genome editing. This requires validation of the target genes, preferably by a nontransgenic DNA-free editing, before the final application. One such important gene regulating sex determination in Drosophila is the Sex lethal (Sxl). However, the function of Sxl is not highly conserved in other insects and, in particular, we are beginning to comprehend its role in Lepidoptera with only one reference available in Spodoptera litura till date. In the present study, we have edited the sxl gene of S. frugiperda through the delivery of ribonucleoprotein complex (sgRNA + Cas9) at G0 stage embryo, targeting the conserved region of all the documented five splice variants. Results clearly showed that editing of sxl gene impacted the overall fecundity and hatching rate. Therefore, Sxl could be one of the target genes for developing pgSIT approach for the management of S. frugiperda.