@article{ec99ee9ffb2f4f2e9d46c295ab384447,
title = "Gene editing of the ABC Transporter/White locus using CRISPR/Cas9-mediated mutagenesis in the Indian Meal Moth",
abstract = "ATP binding cassette (ABC) proteins are involved in transport of substrates across membranes including eye pigments. Mutations of ABC transporter white, brown and scarlet genes of Drosophila and other insects result in visible eye color phenotypes. White locus was identified in a genome assembly of Plodia interpunctella and was found to extend for 16,670 bp comprising 13 exons. We report here recovery of heritable mutants in white in the Indian meal moth, P. interpunctella, using CRISPR/Cas9-mediated mutagenesis. A white eye strain of P. interpunctella c.737delC (Piw-/-) was previously isolated in 1986. Guide RNA (sgRNA) was designed for exon 1 (sgRNA242). Microinjection of Cas9/sgRNA242 complex into Plodia wild type eggs (≤20 min post oviposition) produced 156 viable larvae of which 81 eclosed as adults. Forty-five (56 %) adults displayed wild type phenotype, while 26 females (32 %) and 10 males (12 %) showed full or partial white eye phenotype. The 26 white eye females were mated with Piw-/- males and 21 matings resulted in F1 white eye progeny. Thirteen of the Piw-242 lines were established and sequencing showed indels at the CRISPR/Cas9 242AM site. Based on RT-PCR analysis, most white mutations resulted in suppressed levels of transcript. These results demonstrate the utility of CRISPR/Cas9 gene editing in Plodia which suggests this technology can be used to characterize the role of various genetic elements including those that encode novel targets or confer insecticide resistance mechanisms.",
author = "Shirk, {Bryce D.} and Shirk, {Paul D.} and Furlong, {Richard B.} and Scully, {Erin D.} and Ke Wu and Siegfried, {Blair D.}",
note = "Funding Information: We thank Kathy Leonard and Dr. Jim Campbell at USDA-ARS Stored Product Insect and Engineering Research Unit for rearing and providing the insects, Dr. Scott Geib at USDA-ARS, Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center for advice on assembly and sequencing, and Kris Hartzer for laboratory assistance. Library preparation and sequencing were performed at HudsonAlpha Genomic Services Lab (Huntsville, AL). We also thank Karen Garren for her work to isolate the P. interpunctella Piw- strain. Financial support for BDS was provided through the University of Florida Genetics Institute Seed Grant awarded to Akito Kawahara, Principal Investigator. Funding Information: We thank Kathy Leonard and Dr. Jim Campbell at USDA-ARS Stored Product Insect and Engineering Research Unit for rearing and providing the insects, Dr. Scott Geib at USDA-ARS, Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center for advice on assembly and sequencing, and Kris Hartzer for laboratory assistance. Library preparation and sequencing were performed at HudsonAlpha Genomic Services Lab (Huntsville, AL). We also thank Karen Garren for her work to isolate the P. interpunctella Piw- strain. Financial support for BDS was provided through the University of Florida Genetics Institute Seed Grant awarded to Akito Kawahara, Principal Investigator. The use of trade name, commercial product, or corporation in this publication is for the information and convenience of the reader and does not imply an official recommendation, endorsement or approval by the U. S. Department of Agriculture or the Agricultural Research Service for any product or service to the exclusion of others that may be suitable. Publisher Copyright: {\textcopyright} 2022",
year = "2023",
month = mar,
doi = "10.1016/j.jinsphys.2022.104471",
language = "English (US)",
volume = "145",
journal = "Journal of Insect Physiology",
issn = "0022-1910",
publisher = "Elsevier Limited",
}