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%
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.