|PAULO, DANIEL - University Of Hawaii|
|SIMMONDS, TYLER - Oak Ridge Institute For Science And Education (ORISE)|
Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/3/2022
Publication Date: 10/20/2022
Citation: Paulo, D., Cha, A.Y., Kauwe, A.N., Curbelo, K.M., Corpuz, R.L., Simmonds, T.J., Sim, S.B., Geib, S.M. 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. Journal of Economic Entomology. 115(6):2110-2115. https://doi.org/10.1093/jee/toac166.
Interpretive Summary: Targeting mutations in insect genomes is an established technology through the use of CRISPR/Cas9 mutagenesis. This involves injecting the embryos of a species with a cocktail of components that direct cleavage of the genome in a specific site, and imperfect repair to introduce a mutation that can disrupt a genome or delete a small portion of the genome. Here we describe improvements to previously published CRISPR techniques which increased the mutagenesis rate an order of magnitude, and demonstrate CRISPR-based deletions in the white gene in Zeugodacus cucurbitae, the melon fly, which previously could not be transformed using previously established methods. Finally, we summarize published methods across Tephritidae to provide a global exploration of techniques and methodologies which may lend themselves to being the most productive for generation of positive mutations.
Technical Abstract: Tephritid fruit flies are among the most invasive and destructive agricultural pests worldwide. Over recent years, the family has gained particular attention in genetic manipulation studies using the CRISPR/Cas9 genome-editing system for the development of new strains engineered to facilitate their management and control. However, available protocols for gene knockout in tephritids often result in low mutagenesis/inheritance efficiencies, limiting the potential of the technology to a few of the most thoroughly studied species within the family. Here we propose a simple, reliable, and easy-to-transfer CRISPR/Cas9 targeted gene disruption protocol for in vivo functional analysis of candidate loci in non-model tephritid fruit flies. We demonstrated the efficiency of our protocol by disrupting the white eye (we) orthologue in the melon fly, Zeugodacus cucurbitae. High rates of somatic mosaicism were induced by microinjecting pre-assembled Cas9-sgRNA ribonucleoprotein complexes (RNPs) into non-dechorionated melon fly embryos during early embryogenesis. Adult mosaic flies efficiently transmitted their modified alleles to the subsequent generation with high germline transmission rates, allowing the rapid development of stable mutant strains. We achieved similar results when targeting the we gene in the Oriental fruit fly, Bactrocera dorsalis, using the same experimental design and sgRNA, illustrating the reproducibility of our methods. To our knowledge, 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.