Progress 09/01/24 to 08/31/25
Outputs
Target Audience:Regulators. Scientists who regulate GMOs are a target audience. The PD attends the annual PD directors meeting when it is held. The PD directors meeting is usually well attended by regulators. The PD also discusses our research with regulators that attend major conferences such as the annual meeting of the entomological society of America. Stakeholders. For this project, USDA-APHIS is an important stakeholder. The PD met regularly with USDA-APHIS scientists and staff based in Salinas, CA. We planned a large cage field trial withaD. suzukii fsRIDL strain (FL19) and sought regulatory approval from APHIS BRS. Approval was obtained in summer 2025 and the cage trials had begun at the end of the reporting period. There were 8 cages in total each holding 5 strawberry plants. Funding for the cage trials had been obtained from APHIS. Berry and cherry growers are important stakeholders as they would benefit from any tools for better control of the pest. The PD does not have an extension appointment but does communicate regularly with extension faculty at NCSU who do work with small fruit growers. In the past the PD has given presentations at national and local meetings that were attended by growers. In an effort to reach a wider community interested in gene drive we regularly participate in the weekly meetings of the NC State University Genetic Engineering and Society center. The meetings alternate between in person and virtual. The in person meetings are well attended by faculty and students from the social and biological sciences. The virtual meetings are open more widely and often include people from other states and other countries. The PD and Dr Yadav (the postdoctoral fellow on the project) have given presentations to this group on our gene drive work in D. suzukii. Scientists. We attend and give presentations at scientific conferences as described in "how the results were disseminated". Results from from this research are submitted for publication in peer-reviewed journals. Graduate and undergraduate students. The PD teaches a class every other fall on "Genetic Pest Management" that is mostly taken by entomology graduate students. The PD also taught a module in a Genetics and Genomics overview class that is taken each fall by graduate students from many disciplines. The module is on genetic pest management but with a particular focus on gene drive. Mr O'Brien and Ms. Tarrand co-taught an undergraduate class entitled "Taming Selfish DNA" in spring 2025. The class includes material on gene drive. Media. There has been a sharp increase in interest in the media on New World screwworm with the imminent threat to US agriculture. On several interviews the PD spoke about how genetic suppression using fsRIDL or homing gene drive could be much more efficient than releasing radiation sterilized males and females. General public. In addition to speaking with the media we participate in outreach events to inform the general public about genetic biocontrol in general and research in the Scott lab in particular. Changes/Problems:Dr Yadav spent most of the reporting period completing experiments for another BRAG project (2021-33522-35341). By the end of the reporting period, he was working full time on this project. At the end of the reporting period, the co-PI Dr Anders Huseth resigned from NC State University as he had accepted a position at Michigan State University. Dr Huseth continues to advise on the research with D. hydei and remains a member of Ms. Tarrand's doctoral committee. What opportunities for training and professional development has the project provided?The scientists working on this project are Dr Amarish Yadav and two PhD students, Ariel Tarrand and Casey O'Brien. They attended weekly departmental seminars and weekly genetic and genomics academy (GGA) seminars at NC state. They also attended the annual GGA retreat. They also attended meetings and gave presentations as listed below in how results were disseminated. How have the results been disseminated to communities of interest?Stakeholders. The PD collaborates with USDA-APHIS scientists planning and testing D. suzukii strains in large field cages in Salinas, CA. Letters of support for the field cage trials were obtained from major berry growers in California such as Driscoll's. A field cage trial of the fsRIDL strain was underway at the end of the reporting period. The PD does not have an extension appointment but does communicate regularly with extension faculty at NCSU who do work with small fruit growers. In the past the PD has given presentations at national and local meetings that were attended by growers. Scientists. Invited presentations by the PD: · Leveraging Insect Sex Determination Systems for Genetic Biocontrol of Pests. Department of Biochemistry, Virginia Tech, Blacksburg, VA, February 17, 2025. · Homing gene drive strains for suppression of spotted wing drosophila populations. Keystone Symposia on "Future of Agriculture and Sustainability", Keystone, CO, January 20-23, 2025. Ms. Tarrand attended the Southeastern branch meeting of the Entomological Society of America held in Baton Rouge, Louisiana and gave a presentation entitled "Developing gene drives for the suppression of pest Drosophila" on March 10, 2025. Graduate and undergraduate students. The PD taught the "Genetic Pest Management" class in fall 2024.Thisis a graduate-level class that covers all aspects of genetic biocontrol from sterile insect technique to gene drive . The PD also taught a module in a Genetics and Genomics overview class that is taken each fall by graduate students from many disciplines. The module is on genetic pest management but with a particular focus on gene drive. Media. As listed under stakeholders, the PD gave numerous interviews with reporters over the last year discussing issues related to the current screwworm outbreak and participated in the "talking biotech" podcast. General public. The Scott lab actively participates at the annual "Bugfest" in Raleigh, which draws more than 20,000 people. In 2024, bugfest was held on September 14 in the North Carolina Museum of Natural Sciences and surrounding streets. Our display includes hands-on activities demonstrating gene editing, a poster on gene drive, activities for children (e.g. "find the fly") and videos we produced on genetic biocontrol that were shown on a large TV next to the display. Our table was manned continuously throughout the day by the PD, students, staff and postdoctoral fellows. A highlight in 2024 was that our display was selected to occupy a prominent position near the entrance to the museum, which meant we had many visitors throughout the day from 9am until 7pm! What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will extend the development and evaluation of homing gene drives to the remaining species in the project and continue improving drive designs in D. suzukii. We will also carry out additional cage trials and collect biological data needed for refined population models. Objective 1: Develop homing gene drives in D. hydei and C. macellaria We will design and test guide RNAs (sgRNAs) that target the doublesex gene in both species, first using laboratory assays and then by generating edited flies. The most effective sgRNA for each species will be used to create a fully autonomous, dominant female-sterile drive, inserted using CRISPR/Cas9. If direct insertion of the full drive construct proves inefficient, we will use a two-step backup strategy: first inserting a small marker gene flanked by inverted attP sites, followed by site-specific integration of the full drive construct using phiC31 integrase. Drive efficiency will be assessed by crossing drive males to wild females and measuring inheritance of the marker gene. Objective 2: Improve and evaluate D. suzukii drive systems We will measure drive performance of the autonomous D. suzukii system under temperature conditions that mimic early season (17°C) and mid-summer (25°C). Because recessive-drive males showed lower competitiveness in the cage experiments--contrary to earlier bottle tests--we will modify the drive allele to improve male fitness. The revised design will enhance female-specific splicing using multiple TRA/TRA2 binding sites and retain degron sequences to prevent DSX function in females while minimizing impacts on males. Additional cage suppression trials will be conducted using continuous populations. These will include both: • split-drive males, and • males carrying the fully autonomous dominant female-sterile drive. This will allow us to compare the performance of different drive configurations under realistic conditions. Objective 3: Collect biological data for improved gene drive models We will continue gathering key life-history measurements, including lifetime female fecundity for both D. suzukii and D. hydei, as well as longevity data for D. hydei males. These data will help refine model assumptions and improve predictions. Objective 4: Modeling for cage and future larger-scale evaluations The results from upcoming cage-suppression experiments will be incorporated into our models. We will develop a more detailed, multi-stage model for D. suzukii, suitable for simulating larger populations and more complex environments. We will also begin examining how spatial structure--such as increased cage size or uneven resource distribution--may influence the spread of the drive. For D. hydei, the model will be updated to reflect its specific life-history traits and to explore how vertical spatial structure, such as that found in warehouses or storage areas, could affect population dynamics and gene drive performance.
Impacts
What was accomplished under these goals?
1. Relevance Spotted-wing drosophila (D. suzukii) is a serious invasive pest of soft-skinned fruits and causes major crop losses across the U.S. Current insecticides often fail because they wash off in rain or cannot be used close to harvest. Drosophila hydei is an emerging pest of stored sweetpotato and Cochliomyia macellaria is used as model for the major livestock pest, C. hominivorax. New genetic approaches--such as homing gene drives--may offer a more targeted, sustainable way to suppress populations. However, before any field-ready system can be considered, we must understand how well gene drive systems work in the laboratory, how fast they spread, whether resistance forms, and how they perform under realistic population conditions. This project aims to generate that foundational knowledge. 2. Response: Progress Toward Each Objective Objective 1. Create homing gene drive strains in D. suzukii, D. hydei, and C. macellaria and assess drive efficiencies. During this first reporting period we focused on D. suzukii; work in D. hydei and C. macellaria will occur in Years 2-3. A major challenge for gene drive systems is the possibility that DNA repairs made by the cell will create mutations that block Cas9 cutting, allowing a functional gene to persist and the drive to fail. To reduce this risk, we built and tested drive constructs that use two or three single guide RNAs (sgRNAs) targeting the female-specific exon of the doublesex (dsx) gene. Using two strategies--multiple promoters or tRNA-mediated processing--we created drive lines that produce two gRNAs. In addition, dsx drive lines were established that produce three sgRNAs from one transcript using tRNA processing. Drive constructs were inserted into the doublesex locus using CRISPR/Cas9. Drive females and males were crossed to wild type flies, and offspring were scored for inheritance of a visible fluorescent marker. If no drive occurs, 50% of offspring inherit the marker; perfect drive results in 100%. Key findings: • Two-gRNA systems produced strong drive in males (≈85% inheritance), slightly lower than the best single-gRNA drive (94-99%) but still highly effective. • Drive was lower in females (≈60%), partly because one of the sgRNAs cuts less efficiently in females. • The distance (50 bp) between cut sites may make homology-directed DNA repair less efficient in female germlines. • Three-gRNA systems produced only ~55-60% inheritance in both sexes, likely due to poor sgRNA production through tRNA processing. Based on these findings, future multi-sgRNA systems will use individual promoters for each sgRNA rather than tRNA processing. We also made a dominant female sterile fully autonomous drive, combining Cas9 and the best-performing gRNA (gRNA3) in one construct. Drive efficiency was 89.5%, slightly lower than the split-drive version. When additional Cas9 was supplied genetically, inheritance increased to 97%, showing that Cas9 expression from the autonomous construct is somewhat limiting. Even so, the autonomous system performed strongly and provides a promising foundation for future suppression strategies. Objective 2. Evaluate ability of gene drive strains to suppress laboratory cage populations. Modeling predicted that both dominant and recessive sterile split-drive strains could suppress populations with low release ratios (as low as 1 drive male to 4 wild males). We tested this in four-replicate small-cage trials. Key findings: • Dominant female-sterile drive: A 1:4 release ratio eliminated all populations within nine generations. • Recessive sterile drive: Required doubling the release ratio mid-experiment; elimination occurred after 14 generations. Subsequent tests showed recessive-drive males were outcompeted by wild males, possibly because the optimized splice acceptor used in the construct interfered with male dsx function. We next tested the dominant sterile drive in larger, continuously breeding cages, using a set diet-replacement schedule. Diet was supplied in small petri dishes four times a week. One of the dishes was left in the cage for 2 weeks to replenish the population and another was removed after 24 h to obtain a count of daily egg production. The other dishes were only to provide a food source for adults and were removed after a few days. A 1:1 release ratio sharply reduced populations within 4-5 weeks. All treatment populations were eliminated within ten generations, while control cages persisted. These results show that doublesex-targeting gene drive males can reliably suppress D. suzukii populations in small lab cages. Objective 3. Collect biological data for improved modeling (life-history traits, competitiveness, environmental factors). We measured longevity of drive males (dominant and recessive systems) at 22°C using replicated vial assays. Survival was similar to wild type, indicating no major fitness deficits. We also tested whether larval density dependence affects survival--a factor known to strongly influence mosquito gene drive models. Using densities of 200, 400, 600, and 800 eggs per diet plate (the same system used in the continuous-cage trials), we measured numbers of emerging males and females. Across four replicates there was no significant difference in survival. Therefore, density dependence can be excluded from our D. suzukii models under these experimental conditions. Objective 4. Develop models to predict outcomes in large field cages and confined field settings. We incorporated all laboratory data into updated gene drive models. Major activities: • Estimated key parameters from lab results (drive rate, fecundity, competitiveness). • Parameterized models to reproduce observed small- and large-cage population trajectories. • Built an updated stochastic model for D. suzukii cage trials that captures randomness in survival and mating. • Incorporated improved estimates of age-specific fecundity and newly identified fitness costs. • Used these models to predict optimal release ratios and experimental designs for future cage and semi-field trials. 3. Outcomes (What the project accomplished this year) Across the first reporting year, we: • Developed and tested multi-sgRNA gene drive systems that reduced the likelihood of resistance. • Identified the most effective drive designs and confirmed they can spread efficiently, especially in males. • Demonstrated for the first time that doublesex-targeting gene drives can eliminate D. suzukii populations in both small and large laboratory cage systems. • Collected key biological data (longevity, density dependence) necessary for accurate modeling. • Produced improved population models that now match real laboratory outcomes and guide next-phase experimentation. Together, these outcomes provide the strongest evidence to date that well-designed gene drives can suppress D. suzukii populations under realistic laboratory conditions. 4. Impact (Who benefits and how?) Fruit growers, pest-management professionals, and regulatory agencies are the immediate beneficiaries. This work provides a clearer scientific foundation for evaluating future genetic pest-control tools. By identifying which gene drive designs spread efficiently, which fail, and how populations respond under realistic conditions, this project reduces uncertainty for both researchers and eventual decision-makers. In the long term, these advances may support the development of safer, more effective, and more sustainable control strategies for a damaging invasive pest--potentially reducing crop losses, pesticide use, and production costs.
Publications
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