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ViewAttendees 8

Member Symposium

Farm to Fork: Linking Biology of Postharvest Insects to their Management

7: Gene tagging by CRISPR/Cas9 homology directed recombination in the Indian meal moth, Plodia interpunctella

On-Demand
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ATP binding cassette (ABC) proteins are involved in transport of substrates across membranes. The white, brown and scarlet genes code for ABC transporter proteins that are involved in movement of ommochrome and pteridine precursors into pigment granules in insect eyes. Mutations of ABC transporter genes result in easily observable eye color phenotypes. The White locus was identified and characterized in a genomic DNA assembly of P. interpunctella and the locus extended for 17048 bp comprising 13 exons. Guide RNA (sgRNA) was designed to a target site in exon 1 (sgRNA242). Microinjection of Cas9/sgRNA242 complex into newly laid Plodia wild type eggs produced 26 females (32%) and 10 males (12%) that showed full or partial white eye phenotype (Pi_C9242w-). Twenty-one matings of the females resulted in G1 white eye progeny. Pi_C9242w- lines were subjected to sequencing analysis demonstrating Nonhomologous End Joining (NHEJ) produced insertion and deletion mutations. To conduct CRISPR/Cas9 Homology Directed Recombination (HDR), the Plodia PiD2 cell line was transformed to produce Cas9puro constitutively and then transfected with blocked double stranded DNA that contained homologous matching White242 flanking sequences and an hr5IE1mNeonGreen reporter cassette. mNeonGreen expressing cells were isolated and PCR confirmed the presence of an HDR insert. Co-injection of dsDNA hr5IE1mNeonGreen reporter into embryos resulted in G0 adults with white eyes and expressing mNeonGreen. These studies demonstrate the utility of gene editing to tag selected genes to characterize the role of various genetic elements in Plodia