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ARS Home » Plains Area » Kerrville, Texas » Knipling-Bushland U.S. Livestock Insects Research Laboratory » LAPRU » Research » Research Project #433423

Research Project: CRISPR/Cas9 to Develop Odorant Co-receptor Mutants of the Screwworm: A Perspective on the Evolution of Olfactory Perception in Blowflies

Location: Livestock Arthropod Pests Research

Project Number: 3094-32000-041-01-N
Project Type: Non-Funded Cooperative Agreement

Start Date: Sep 1, 2017
End Date: Aug 31, 2019

Objective 1: Establish and evaluate the CRISPR system for the functional characterization of the co-receptor Orco in screwworms. Objective 2: Develop a stable Orco mutant line of screwworms. Objective 3: Evaluate the ChomOrco protein deficiencies in screwworm and test the mutant line for impaired olfactory responses.

Obj. 1. The ChomOrco gene will be examined for Cas9 target sites (5’-N20NGG-3’). Results will assist the design of single guide RNAs (sgRNAs) to be evaluated for putative off-targets in the draft genome of screwworm as well as other genome sequences of closely related species. Highly specific sgRNAs will be synthesized in the laboratory. Once produced, micro-injection of screwworm embryos will be done with pairs of sgRNAs targeting two sites within the first 720bp of the ChomOrco coding region (domains TM1-TM418) along with the Cas9 protein (final concentration 200:500ng/µl, respectively). The goal is induction of large deletion events between the targeted-regions. Individual genomic DNA samples, from single adult wings or legs of flies resulting from embryo injections (G0 generation), will be analyzed by PCR and electrophoresis for the presence of mosaic mutations induced by Cas9 cleavage and subsequent double-strand DNA break repair by non-homologous end joining. Genotyping will also demonstrate the CRISPR mutagenesis efficiency in the NWS. Obj. 2. Genotyped G0 individuals will be individually backcrossed with wild type flies. Offspring (G1 heterozygous flies) will be screened for the presence of inherited mutations using the individual genotyping method and subsequent sequencing. Heterozygous G1 (Orco+/-) flies carrying larger deletions in ChomOrco will be tested for their founder capacities by a second individual backcrossing to wild type flies. The presence of mutations in G2 flies will be confirmed as described above; lines carrying the heritable mutated alleles will be used to expand the colonies. The Orco-/- individuals will be selected at every generation until the strain is made homozygous (taking at least four generations; about 4 months). Obj. 3. ChomOrco mRNA stability in mutant flies will be evaluated by RT-qPCR. Relative expression analysis will be performed under the comparative Ct method using three replicates, non-template controls and the ChomGAPDH. The localization of ChomOrco protein within the adult antennae will be verified by immunohistochemistry, following previously described protocols, using the primary antibody IC3 (HWYDGSEEAKT, located within the intracellular loop connecting TM6 to TM7) and the Olympus FV1000 laser scanning confocal microscope. Electroantennograms will be used to test for impaired olfactory-mediated responses by ChomOrco mutants; responses to chemical stimuli will be recorded using previously described protocols. Pre-selected compounds and blends were selected that mimic the NWS olfactory-mediated behaviors including: (a) food searching, (b) mating, and (c) host and oviposition site selection. Behavioral observations will be done using the Y-tube olfactometer (adults), Winner Spot (larvae) dual-choice setups, and the Hammack’s glass cage (copulatory response). For comparisons, all analysis will be performed with flies carrying ChomOrco mutated and WT alleles (heterozygotes and homozygotes).