How to translate text using browser tools
20 October 2022 A Unified Protocol for CRISPR/Cas9-Mediated Gene Knockout in Tephritid Fruit Flies Led to the Recreation of White Eye and White Puparium Phenotypes in the Melon Fly
Daniel F. Paulo, Alex Y. Cha, Angela N. Kauwe, Keena Curbelo, Renee L. Corpuz, Tyler J. Simmonds, Sheina B. Sim, Scott M. Geib
Author Affiliations +
Abstract

Tephritid fruit flies are among the most invasive and destructive agricultural pests worldwide. Over recent years, many studies have implemented the CRISPR/Cas9 genome-editing technology to dissect gene functions in tephritids and create new strains to facilitate their genetics, management, and control. This growing literature allows us to compare diverse strategies for delivering CRISPR/Cas9 components into tephritid embryos, optimize procedures, and advance the technology to systems outside the most thoroughly studied species within the family. Here, we revisit five years of CRISPR research in Tephritidae and propose a unified protocol for candidate gene knockout in fruit flies using CRISPR/Cas9. We demonstrated the efficiency of our protocol by disrupting the eye pigmentation gene white eye (we) in the melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae). High rates of somatic and germline mutagenesis were induced by microinjecting pre-assembled Cas9-sgRNA complexes through the chorion of embryos at early embryogenesis, leading to the rapid development of new mutant lines. We achieved comparable results when targeting the we orthologue in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), illustrating the reliability of our methods when transferred to other related species. Finally, we functionally validated the recently discovered white pupae (wp) loci in the melon fly, successfully recreating the white puparium phenotype used in suppression programs of this and other major economically important tephritids. This is the first demonstration of CRISPR-based genome-editing in the genus Zeugodacus, and we anticipate that the procedures described here will contribute to advancing genome-editing in other non-model tephritid fruit flies.

Published by Oxford University Press on behalf of Entomological Society of America 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US.
Daniel F. Paulo, Alex Y. Cha, Angela N. Kauwe, Keena Curbelo, Renee L. Corpuz, Tyler J. Simmonds, Sheina B. Sim, and Scott M. Geib "A Unified Protocol for CRISPR/Cas9-Mediated Gene Knockout in Tephritid Fruit Flies Led to the Recreation of White Eye and White Puparium Phenotypes in the Melon Fly," Journal of Economic Entomology 115(6), 2110-2115, (20 October 2022). https://doi.org/10.1093/jee/toac166
Received: 4 August 2022; Accepted: 3 October 2022; Published: 20 October 2022
JOURNAL ARTICLE
6 PAGES

This article is only available to subscribers.
It is not available for individual sale.
+ SAVE TO MY LIBRARY

KEYWORDS
CRISPR/Cas9 genome-editing
functional genomics
tephritid fruit fly
white eye
white pupae
RIGHTS & PERMISSIONS
Get copyright permission
Back to Top