Gene editing of the ABC Transporter/White locus using CRISPR/Cas9 mutagenesis in the Indian Meal Moth

Authors: SHIRK, BRYCE - University Of Florida; Shirk, Paul; Furlong, Richard; Scully, Erin; WU, KE - University Of Florida; SIEGFRIED, BLAIR - University Of Florida

Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2022
Publication Date: 12/7/2022
Citation: Shirk, B.D., Shirk, P.D., Furlong, R.B., Scully, E.D., Wu, K., Siegfried, B.D. 2022. Gene editing of the ABC Transporter/White locus using CRISPR/Cas9-mediated mutagenesis in the Indian meal moth. Journal of Insect Physiology. 145:104471. https://doi.org/10.1016/j.jinsphys.2022.104471.
DOI: https://doi.org/10.1016/j.jinsphys.2022.104471

Interpretive Summary: The acquired pesticide resistance is a major limitation to utilization of many currently registered pesticides including Bt toxins employed in bioengineered crops. Understanding the genetics of resistance in the pest insects requires being able to have mutations in these critical genes. Researchers at the USDA-ARS, Center for Medial, Agricultural and Veterinary Entomology, Gainesville, Florida, USDA-ARS Manhattan, Kansas, and the University of Florida collaborated to create mutations in genes that produce transport proteins in the Indian meal moth using gene editing. Gene editing of specific transporter genes cause mutations in a protein that produced white eyed moths and effected Bt resistance. Using this gene editing system, various pesticide resistance genes can be mutated and the effects studied in order to find mechanisms to avoid or reverse pesticide resistance.

Technical Abstract: The superfamily of ATP binding cassette (ABC) proteins is involved in transport of substrates across membranes. Transport of substrates is important to normal cellular functions such as formation of eye pigments. However, the ABC transporters can also be inadvertent partners of toxins, such as Bt Cry1A toxin, that poison the cell. The white, brown and scarlet genes code for ABC transporter proteins involved in movement of ommochrome and pteridine pathway precursors into pigment granules in the eye. Mutations in ABC transporter genes result in eye color phenotypes that are easily observed. This research examines the role of the white/ABC transporter in moths by using CRISPR/Cas9 gene editing. A white eye strain of the Indian meal moth, Plodia interpunctella, (Piw-) was isolated and has been in culture since 1992. The white locus was identified and characterized in a genomic DNA assembly of P. interpunctella (Scully and Oppert, unpublished). White extended over 17048 bp and was comprised of 13 exons. Using these genomic sequences, guide RNAs (sgRNA) were designed to target sites in exon 1 (sgRNA242). G0 adults with the white eye phenotype were recovered from adults from embryos that were microinjected with the Cas9/sgRNA242. Following mating of white eye sgRNA242 females with Piw- males, multiple strains were established. Deletions at the target site for sgRNA242 were recovered and characterized. Deletions resulted in truncation of the transcript or produced read through that resulted in defective proteins.