Source: UNIV OF CALIFORNIA-SAN DIEGO submitted to NRP
DEVELOPMENT OF TECHNOLOGIES AND PRACTICES TO MONITOR AND MINIMIZE THE RISK OF GENETIC TECHNOLOGIES FOR THE CONTROL OF AGRICULTURAL PESTS.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1032497
Grant No.
2024-33522-42695
Cumulative Award Amt.
$649,414.00
Proposal No.
2024-03787
Multistate No.
(N/A)
Project Start Date
Sep 1, 2024
Project End Date
Aug 31, 2028
Grant Year
2024
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
UNIV OF CALIFORNIA-SAN DIEGO
9500 GILMAN DRIVE
LA JOLLA,CA 92093
Performing Department
(N/A)
Non Technical Summary
The spotted wing Drosophila, Drosophila suzukii, is a worldwide crop pest of soft-skinned fruits. Current methods to control many agricultural pests, such as D. suzukii, rely on expensive, broad-spectrum insecticides, which have variable efficacy, are challenging to use due to the timing of fruit infestation and increase the risk D. suzukii evolving resistance to the insecticide. However, there are no practical alternatives to managing D. suzukii infestation, and it is likely that, unless more effective control measures are developed, this pest will continue to spread and negatively impact fruit production.Genetic technologies can complement existing pest management strategies for D. suzkuii and other agricultural pests. This proposal aims to generate tools to monitor, improve, and better understand the behavior of a new genetic technology, precision-guided sterile insect technique (pgSIT), and other CRISPR-based technologies in the field. The first tool, Sensitive Enzymatic Nucleic Acid Sequence Reporter (SENSR), is a diagnostic system designed to rapidly detect pgSIT-specific and CRISPR-associated DNA. Rapid identification of the DNA of these technologies is vital for assessing the risk and performance of genetic biocontrol tools. They can be used as a rapid tool to monitor the behavior of genetically engineered (GE) technologies in the field and their potential spread into nearby areas.Another technology we aim to develop in D. suzukii is SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter). SEPARATOR can improve the quality and accuracy of field releases of D. suzukii. For example, only D. suzukii females cause damage to fruit, so we want to reduce crop damage we only want to release males in genetic control programs. Additionally, if there are frequent errors in the sex-sorting of pgSIT lines prior to mating in the laboratory, then there is a higher risk of releasing fertile flies. We will, therefore, engineer and optimize a SEPARATOR system in D. suzukii that should minimize or eliminate the risk of mating errors that might result in the release of females that damage crops or fertile individuals.We will also evaluate the behavior of pgSIT in response to changes in environmental conditions and whether pgSIT could help mitigate the gene flow or spread of gene technologies. These studies will ensure we understand how pgSIT will perform in a changing environment and how it can be used to remove other GE technologies from the field. These studies will be performed in the laboratory and modeled to understand how they will behave on a larger scale.Studies of these emerging technologies are essential for the responsible development and management of genetically engineered (GE) technologies for agricultural pest control. This work will focus on a more comprehensive evaluation of the CRISPR-based SIT technology, pgSIT, for D. suzukii control and technologies that complement its monitoring and success in the field. This will allow us to build tools and knowledge to suppress pest populations and better confine and manage more invasive genetic technologies.
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
(N/A)
Developmental
80%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110108050%
2113110310050%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1080 - Genetics; 3100 - Management;
Goals / Objectives
Objective 1: Development and assessment of a rapid GE monitoring tool for agricultural pests. Objective 1 (Monitor dispersal- Program Areas 1 and 2) will evaluate a CRISPR-based surveillance tool to monitor the persistence of the pgSIT genetic elements in agricultural pests. This objective focuses on evaluating a transgene detection technology to aid in monitoring performance and risk assessments of synthetic technologies in the field. We have recently engineered multiple diagnostic platforms based on the CasRx, an RNA-targeting protein (Sensitive Enzymatic Nucleic-acid Sequence Reporter (SENSR). We will evaluate this technology in population cages to identify synthetic elements and prepare them for field monitoring. This objective will be divided into the following tasks: Task 1.1: Engineer and validate SENSR technology for the detection and monitoring of pgSIT technologies in D. suzukii; Task 1.2: Validate the SENSR technology for the detection and monitoring of genetic and gene drive technologies that utilize the CRISPR-Cas9 technology.Objective 2: Development of a sex-sorting technology to improve the accuracy and predictability of GE field releases. Objective 2 (Improve the management and release practices- Program Areas 1 and 2) will develop a precise sex-sorting technology, SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) in the agricultural pest, D. suzukii. SEPARATOR can improve the quality and accuracy of released GE D. suzukii. The accuracy of sex sorting technologies in genetically engineered (GE) release programs can impact the success of the program and the safety of agricultural commodities. This objective will be divided into the following tasks: Task 2.1: Validating female-specific expression of the fluorescent protein in D. suzukii; Task 2.2: Validating the SEPARATOR technology in pgSIT D. suzukii.Objective 3: Understanding environmental impacts on the ecology and behavior of GE insects. Objective 3 (Understand the ecology and behavior of GE organisms in response to environmental conditions- Program Areas 2 and 4) will evaluate the fitness of pgSIT in response to different environmental conditions expected in the field to understand key environmental factors that may impact its behavior. This objective will be divided into the following tasks: Task 3.1: Impact of environmental factors on pgSIT population behavior; Task 3.2: Impact of environmental factors on pgSIT-SEPARATOR population behavior.Objective 4: Evaluating pgSIT as a tool for managing invasive genetic technologies. Objective 4 (Mange/Mitigate the spread of more invasive genetic technologies- Program Area 1) will evaluate the long-term stability of pgSIT as a mitigative strategy to prevent the unintended spread of other genetic technologies. This objective will determine whether pgSIT technology can be used for removing gene drive systems from populations. Currently, we have both threshold-dependent and independent gene drive populations of D. suzukii in our laboratory, which we will use to study whether pgSIT technology can support removing these drives from laboratory populations. This objective will have the following task: Task 4.1: pgSIT for removing threshold-dependent Medea drives from D. suzukii populations.
Project Methods
Objective 1: SENSR assays will be designed and performed using a two-step nucleic acid detection protocol. Target sequences will be amplified in isothermal preamplification reactions using recombinase polymerase amplification (RPA). The in vitro transcription will be coupled with the cleavage assay and a fluorescence readout using 6-carboxyfluorescein (6-FAM) and then immediately run on a LightCycler 96 at 37°C for 60 minutes using our standard acquisition protocol. We will generate multiple gRNAs to target ≥ four target regions within each D. suzukii gRNA transgene or Cas9 to assess the SENSR detection capabilities. The gRNAs and Cas9 targets that have high sensitivity and specificity at the lowest dilutions will be evaluated further in a multiple-pool, multiple-line SENSR assay. This will allow us to develop and validate a SENSR system to specifically detect D. suzukii pgSIT flies.Objective 2: To engineer the female-specific expression of a reporter for positive selection of females, we will exploit the sex-specific alternative splicing of a conserved sex-determination gene, traF. We will generate two sets of dual fluorescent marker constructs encoding a fluorescent marker for both sexes and a female-specific fluorescent marker and integrate these constructs in D. suzukii with piggyBac-mediated embryo injections. Transgenesis will be screened by a fluorescent marker, and lines will be crossed and expanded. A comprehensive analysis utilizing reverse transcription polymerase chain reaction (RT-PCR) and RNA sequencing will be conducted to validate the sex-specific splicing pattern of the traF splicing modules. We will evaluate the intensity and sex specificity of the fluorescent reports over multiple life stages. At the egg, larval, and pupa stages, the percentage of individuals with the correct sex-specific fluorescence and their fitness will be determined. Transgenic lines with consistently accurate and bright sex-specific fluorescent markers during early development stages and that have high fitness will be selected for integration into one or more Cas9 and gRNA pgSIT D. suzukii lines. The life stage sex-specific fluorescence and fitness of the Cas9-SEPARATOR and gRNA-SEPARATOR systems will also be evaluated, and we will also study them in small population studies and by population modeling.Objective 3: The parent pgSIT or pgSIT-SEPARATOR Cas9 and gRNA lines, their F1 pgSIT transheterozygote progeny, and wt flies will be raised at standard conditions and at varied temperatures and humidity. These groups will then be assessed for their fitness and mating competitiveness. If there are significant differences in fitness and mating competition between any temperature/humidity variation and the controls, additional population suppression and modeling experiments will be performed to determine the impact of temperature/humidity on pgSIT populations of D. suzukii. We will also evaluate target reduction and transgene and global expression for significantly different conditions.Objective 4: We will perform several long-term multigenerational population cage experiments to determine how pgSIT can call back a gene drive system. We will seed populations with a Medea drive by introducing Medea-bearing fathers and WT strain mothers at high introduction frequencies. The Medea drive will be driven to >80% frequency in each population. Populations that maintain this >80% frequency (proportion of individuals with a Medea element) for ≥5 generations will then have pgSIT sterile males released into their populations at 1:2, 1:3, and 1:5 ratios of Medea/wt individuals to pgSIT sterile males. Controls with only wt and Medea releases will also be evaluated. We will release these ratios each generation and monitor the population size and gene drive frequency over multiple generations. For the Medea drive modeling, we will use the mathematical modeling we used to develop the drive to compare control and pgSIT-treated populations. Model fitting will be carried out using Bayesian Markov chain Monte Carlo (MCMC) methods, and parameters describing the population dynamics of the Medea drive will be estimated.

Progress 09/01/24 to 08/31/25

Outputs
Target Audience:The target audiences include the scientific community, organizations, and individuals interested in controlling Drosophila suzukii. The tools we develop are useful for the study, control, and scaling of D. suzukii production, and these technologies have direct applications to the development of control technologies for this important agricultural pest. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided extensive training opportunities in both technical and professional domains for early-career researchers. On the technical side, the work has enabled the development of advanced molecular biology skills, including the design of CRISPR/Cas9 constructs, gRNA validation, and gene expression analysis through RT-qPCR and sequencing. Researchers have gained experience in transgenic insect husbandry under various environmental conditions, as well as in phenotypic assays such as fertility testing, identification of intersex phenotypes, and developmental analysis. Additional training has included data management and statistical analysis, microscopy techniques, and the optimization of experimental design for inducible gene expression systems. Beyond technical expertise, the project has supported broader professional development. Team members have contributed to scientific writing for publications and reports, presented findings at meetings, and engaged in interdisciplinary collaboration between molecular biology, entomology, and applied pest management. The project has also provided training in mentoring of junior students, strengthening leadership and communication skills. Collectively, these opportunities have prepared researchers for future independent work while enhancing their competitiveness for academic, government, and industry career pathways. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Genetic technologies can complement existing pest management strategies for D. suzkuii and other agricultural pests. This proposal aims to develop tools to monitor, improve, and enhance the understanding of the behavior of precision-guided sterile insect technique (pgSIT) and other CRISPR-based technologies in the field. Objective 1: Development and assessment of a rapid GE monitoring tool for agricultural pests. We have collected some D. suzukii samples; however, in the next reporting period, we will begin evaluating these samples using the SENSR assay. Candidate gRNAs will be evaluated for sensitivity and specificity across varying ratios of transgenic to wild-type flies, with the best-performing guides advanced to pooled assays. In parallel, Cas9 detection targets originally developed for Aedes aegypti will be validated in D. suzukii to determine whether the SENSR system can reliably identify Cas9 transgenes across species. Together, these experiments will establish and validate a SENSR-based platform for rapid and specific detection of pgSIT transgenes in agricultural pests. Objective 2: Development of a sex-sorting technology to improve the accuracy and predictability of GE field releases. Our next step is to engineer a dual pgSIT and SEPARATOR system that can be used to scale D. suzukii production. We will then test the fitness and performance of this dual system under different environmental conditions. We will also continue to develop the temperature inducible female killing system, which can also be used to scale D. suzukii production in control programs. In the next reporting period, we plan to engineer more lines and test them under numerous heat shock protocols to optimize these systems to kill or sterilize females. Objective 3: Understanding environmental impacts on the ecology and behavior of GE insects. Once we develop the dual pgSIT-SEPARATOR lines, the parent pgSIT and pgSIT-SEPARATOR Cas9/gRNA lines, their F1 pgSIT transheterozygote progeny, and wild-type flies will be reared under standard (21°C) and variable temperature (16°C, 26°C) and humidity (10-80%) conditions. These groups will be evaluated for fitness, longevity, and mating competitiveness through assays measuring courtship success, egg laying, and hatching rates when pgSIT and wild-type males compete for wild-type females. For lines showing significant fitness or competitiveness changes under altered environments, additional analyses will assess transgene target reduction, Cas9/gRNA expression, and global expression profiles. Population suppression experiments will then be performed under both control and variable conditions, with outcomes incorporated into population modeling to predict pgSIT and pgSIT-SEPARATOR performance under environmental stress. Five to ten biological replicates will be used to ensure statistical robustness. Objective 4: Evaluating pgSIT as a tool for managing invasive genetic technologies ?We will test whether pgSIT sterile male releases can be used to remove gene drives from D. suzukii populations. We will initiate the long-term, multigenerational cage experiments to establish the gene drive at high frequencies (>80%) and then introduce pgSIT males at varying release ratios (1:2, 1:3, 1:5) to assess their ability to reduce drive prevalence, with appropriate wild-type and drive-only controls. Parallel mathematical modeling, utilizing Bayesian MCMC methods and prior parameter estimates of drive dynamics (fitness costs, resistant allele frequencies, and toxin efficiency), will predict outcomes under various conditions, including susceptible versus resistant populations. Together, experiments and modeling will determine whether pgSIT can effectively "call back" or eliminate Medea drives from established populations.

Impacts
What was accomplished under these goals? Objective 1: Development and assessment of a rapid GE monitoring tool for agricultural pests. We are developing a SENSR system in D. suzukii that can rapidly identify individuals and pools of individuals containing the Cas9 transgene or a pgSIT-specific gRNA. We have developed similar systems in Aedes mosquitoes (Li et al. 2023, eLife), and are taking this approach to identify transgenes in D. suzukii. In the field, the abundance of the pest (# of flies of the target species/number of trapping events), and the number and percentage of pools positive for these transgenes (# of transgene-positive pools/total number of pools test x100) can be used the develop maximum likelihood estimates (MLE) of the prevalence of the transgene in the population or the transgene (the average number of target species collected per trapping period x the proportion of target species with the transgene). These key surveillance metrics can allow us to rapidly and accurately monitor the abundance, spread, and persistence of CRISPR-Cas9 and pgSIT in the environment. This SENSR monitoring system and the data collected in this project will facilitate the development of a workflow and analysis pipeline to rapidly identify and assess Cas9 and pgSIT transgenes in a population. Objective 2: Development of a sex-sorting technology to improve the accuracy and predictability of GE field releases. We evaluated the potential of SEPARATOR, a fluorescence-based sex-sorting system, for sex-sorting D. suzukii, which will facilitate its large-scale production. We engineered three SEPARATOR constructs containing female-specific transformer (traF) introns from Ceratitis capitata (CctraF), Drosophila melanogaster (DmtraF), and D. suzukii (DstraF). In all strains, only females expressed dsRed alongside eGFP, while males expressed only eGFP, resulting in 100% sorting accuracy across approximately 1,200 flies per strain over 26 generations. Female-specific dsRed expression was first detectable at the second instar larval (L2) stage, enabling early-stage sex identification. Among the constructs, the CctraF intron produced the strongest fluorescence, suggesting it may be the most efficient option for practical implementation of SEPARATOR for D. suzukii. We also evaluated the fitness traits of these lines, including embryo hatch rates and larval-to-adult recovery, and found no significant differences between the SEPARATOR strains, the wild type, and the control transgenics (p < 0.05). This indicates that SEPARATOR does not impose a measurable fitness cost, an important factor for large-scale rearing. Taken together, these results demonstrate that SEPARATOR can provide reliable, efficient, and scalable sex separation in D. suzukii, offering a powerful tool for enhancing sterile insect technique (SIT) programs by reducing rearing costs, preventing the release of sterile females, and improving overall suppression efficiency. While pgSIT can generate sterile males, it is limited by the need for separate Cas9/gRNA strains, sex sorting, and high rearing costs. So, we are also building conditional systems that only allow Cas9 expression under certain conditions. We developed a Cas9 system in D. suzukii that was heat-shock-inducible when driven by the Hsp70Bb promoter. This promoter was selected due to its conservation, chemical-free activation, and compatibility with D. suzukii's thermal tolerance. This heat shock-inducible Cas9 system combined poly-PRE elements with the pDmHsp70Bb promoter and two gRNAs targeting the sex lethal gene (sxl#1, sxl#2) for female-specific lethality. At 18°C, heat shock for 1-4 h at 37°C produced intersex phenotypes in 1.7-12.4% of females, with >40% remaining fertile. At 26°C, a 2 hour heat shock induced intersex phenotypes in 46.3% of adults, but only 1.6% were sterile females. RT-qPCR confirmed inducible Cas9 expression post-heat shock, though low basal expression was detected at non-inducing temperatures. Fitness assays showed no significant reduction in male fertility, mating competitiveness, or longevity under the tested conditions. While PREs reduced leaky expression, suppression was incomplete, suggesting that further refinement is required to achieve full penetrance of female lethality and minimize background Cas9 activity. Objective 3: Understanding environmental impacts on the ecology and behavior of GE insects. We have not started studies with the parent pgSIT/pgSIT-SEPARATOR lines, their F1 transheterozygotes, and wild-type flies under varying temperature and humidity to assess fitness and mating competitiveness. We plan to start these studies during this project period. When these results are finalized, conditions causing significant differences will be further tested through population suppression modeling, alongside evaluation of target reduction and transgene/global expression. Objective 4: Evaluating pgSIT as a tool for managing invasive genetic technologies We have not started this objective, but in the next reporting period, we will begin determining whether pgSIT can be used to remove a gene drive from D. suzukii populations in a release ratio-dependent manner and at a faster rate than releasing wildtype D. suzukii alone.

Publications

  • Type: Other Journal Articles Status: Published Year Published: 2025 Citation: Liu, J., Rayes, D., Yang, M. & Akbari, O. S. Fluorescent-based sex-separation technique in major invasive crop pest, Drosophila suzukii. GEN Biotechnol. 4, 2936 (2025).