Source: NORTH CAROLINA STATE UNIV submitted to NRP
GENOME EDITING OF THE THRIPS SUPERVECTOR, FRANKLINIELLA OCCIDENTALIS, USING TRADITIONAL AND NON-TRADITIONAL METHODS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025794
Grant No.
2021-67013-34575
Cumulative Award Amt.
$300,000.00
Proposal No.
2020-05666
Multistate No.
(N/A)
Project Start Date
Aug 1, 2021
Project End Date
Jul 31, 2024
Grant Year
2021
Program Code
[A1191]- Agricultural Innovation through Gene Editing
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Entomology and Plant Pathology
Non Technical Summary
The western flower thrips, Frankliniella occidentalis, is global pest and a vector of tomato spotted wilt virus (TSWV) and related orthotospoviruses that cause significant yield losses in crops. Our long-range goal is to develop genome-editing technologies for population suppression and/or population replacement of F. occidentalis as a means of reducing reliance on conventional chemical management strategies and mitigating orthotospovirus transmission by this supervector. Genome-editing has not yet been applied to thrips and application of these technologies will also enable basic studies of gene function in thrips, including virus-vector interactions. The aim of this proposal is to develop and optimize a proof-of-principle CRISPR/Cas9 gene-knockout system for F. occidentalis using traditional and new methodologies of delivery of Cas9 and guide RNAs. We will target genes with easily scored visible phenotypes which will enable direct feedback thereby hastening progress and clearly signaling success. The specific research objectives are to:Objective 1. Develop methods for microinjecting precellular F. occidentalis embryos.Objective 2. Use the CRISPR/Cas9 system to knockout F. occidentalis eye-color genes.Our research addresses the USDA-NIFA challenge of developing more sustainable, productive and economically-viable plant production systems. Specifically, we will expand the use of gene editing technology to vectors of plant viruses for pest and disease control.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21131101040100%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The aim of this proposal is to develop and optimize a proof-of-principle CRISPR/Cas9 gene-knockout system for F. occidentalis using traditional and new methodologies of delivery of Cas9 and guide RNAs. We will target genes with easily scored visible phenotypes which will enable direct feedback thereby hastening progress and clearly signaling success. The specific research objectives are to:Objective 1. Develop methods for microinjecting precellular F. occidentalis embryos.Objective 2. Use the CRISPR/Cas9 system to knockout F. occidentalis eye-color genes.
Project Methods
Methods - abbreviated:Collection of precellular F. occidentalis embryosUsing a methodology similar to that already used in our labs, F. occidentalis eggs will be collected 3 hours post oviposition by filtering oviposition medium (3% sucrose solution) through a cell culture basket and rinsing with room temperature double-distilled water. Since eggs are normally oviposited within leaf tissue it is likely that F. occidentalis embryos will require high humidity. Therefore we will handle F. occidentalis embryos similar to those of P. maidis which are also normally oviposited within leaves. With the aid of a stereomicroscope for visualization and a fine-tipped paint brush, embryos will be transferred from the wash basket to a strip of double-sided tape affixed to a glass coverslip. To help keep embryos moist while they await microinjection, coverslips with embryos will be placed on a 1% agar plate.Microinjection of precellular F. occidentalis embryosEmbryos will be microinjected using a stereomicroscope-based injection system housed inside a humidified chamber that is currently located in a dedicated room of the NCSU Insectary to ensure that the working environment is near 100% humidity throughout the microinjection process. Embryos will initially be injected through the chorion to deliver picoliter volumes of injection buffer (fourfold dilution of a phenol red solution, Sigma). Following injection, embryos will be held for three days in a humidified chamber at 23°C. During optimization, we will compare hatch rates of buffer injected embryos, to those of control embryos (non-injected and mock-injected), keeping environmental conditions and handling steps similar between the different injection treatments (buffer-injected vs. controls). Mock-injected embryos will be achieved by "poking" with a closed injection needle.Selection of genome editing targetA large number of mutations are known to affect the pigmentation of the compound eye of Drosophila, including those that affect the biosynthesis or transport of ommochrome and pteridine pigments. Eye-color genes such as scarlet, white and brown are each required for proper pigmentation of the fly eye, with white playing a key role in the transport of both ommochrome and pteridine pigments. Given its pivotal role, loss-of-function mutations in white result in a complete loss of eye pigment. However, while mutations in vermilion result in red-eyed flies (hence the name), insects that lack pteridine eye pigments will have white eyes upon loss of vermilion function. Therefore, without prior knowledge of which pathways pigment a species' eyes, white is frequently the first target to be tested. We have already identified genomic scaffolds containing thrips orthologs of several eye-color genes, including white, vermilion and cinnabar, and plan to test white first since it is expected to produce the most dramatic phenotype.A minimum of three guides per gene will be designed to target highly conserved regions of the gene. For example, when designing guides for ABC transporters like white, we aim to interrupt the Walker A or Walker B motifs since these are required for proper function of the protein. Moreover, we have found that using multiple guides is better than one, and that this approach usually results in the loss of the intervening sequence. Guides will be designed using a combination of online freeware - e.g. CRISPOR, and guide RNAs synthesized by our usual provider (Synthego).Preparation of Cas9/guide RNA complexesCas9 nuclease can be provided in many forms (DNA, RNA, or protein), as can the guide sequences (DNA or RNA). We have found that purified Cas9 protein pre-complexed with sgRNAs is most effective. To form the Cas9-RNA complex we simply mix Cas9 protein (Invitrogen TrueCut) and sgRNAs (Synthego) and incubate at room temperature for 30 minutes. For visualization during injection we add phenol red dye (Sigma), then briefly mix and centrifuge to precipitate particles that could clog the needle. Microinjection will be carried out using the methods developed in Obj 1. DNA from a subset of injectees isolated 24 hours post-injection, will give us rapid insight into the presence/absence of Cas9-mediated cleavage. For each set of guides, our standard protocol (Adrianos et al., 2018) will be used to amplify and sequence across the target site. We will determine the frequency of insertions and deletions (indels) using the quantitative online tool TIDE. This is an efficient way of assessing guide-cutting efficiency, thereby allowing us to select the best guides and targets for the remainder of the project.Post-injection rearing and phenotypic screeningFor Cas9-mediated knockout of eye-color genes, injected embryos will be held for ~3 days at 25oC, and late-stage embryos screened for visible phenotypes. Similar to RNAi, gene-editing experiments can produce a range of phenotypes in the injected individuals. Eyespot pigmentation of some injectees are indistinguishable from wild-type, while others display varying degrees of pigmentation. Also, the proportion of injectees exhibiting a mutant phenotype can vary due to 1) accessibility of the RNP complex to the target gene, 2) efficiency of the guide RNAs and 3) the experimenter performing the injections. However, one of the most important factors remains the ability to introduce the RNP complex prior to cellularization. Moreover, the fewer nuclei present at the time of injection, the higher the percentage of gene-edited cells, thereby increasing the importance of injecting the youngest embryos possible.Importantly, thrips have a significant advantage over other insect species for which we have established CRISPR-mediated genome editing in that males are haploid. The fact males only have a single set of chromosomes makes the task of identifying mutations in recessive eye-color genes far easier. Injected individuals will be mass-mated, and their progeny screened for changes in eye pigmentation. While injected individuals will at best be mosaic, having a mix of wild type and mutant somatic and germ-line cells, eye-color mutations will quickly be revealed in their male progeny. Gene-edited strains can either be produced by crossing mutant males to mutant females (biallelic knockout) or potentially mutant females (siblings with wild-type eye color) or to wild-type females. Once sufficient progeny are available, some can be sacrificed for a PCR screen to verify proper editing of the target gene. We will establish homozygous mutant colonies by mass-mating individuals with mutant eye color.

Progress 08/01/21 to 07/31/24

Outputs
Target Audience:Target audiences reached include undergraduate students and the graduate students that participated in the research and received training by the PIs. Research findings were presented to scientists, extension faculty, and stakeholders at local, regional, and national meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Findings from this project were used in lectures and guest lectures in graduate courses (Molecular Plant Microbe Interactions and Vector Transmission of Plant Pathogens at NCSU) to describe current research on vector transmission of plant pathogens and in presentations to scientists and extension faculty. The project has contributed to the training of 2 graduate students, 1 research associate, and an undergraduate researcher. Based on this work, the lead author, Dr. Jinlong Han (PhD Graduate from Rotenberg Lab, NCSU, currently a postdoc at Colorado State University has been invited the present CRISPR-mediated genome editing of Frankliniella occidentalis at the upcoming Plant and Animal Genomics Conference, and to assist Dr. Lorenzen as a workshop organizer. How have the results been disseminated to communities of interest?The research has been presented to scientists, extension faculty, and stakeholders at local, regional, and national meetings, invited departmental seminars and has been published in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Develop methods for microinjecting precellular F. occidentalis embryos. 1. Wrote detailed protocol which was published in Insect Molecular Biology (see doi: 10.1111/imb.12913). Objective 2. Use the CRISPR/Cas9 system to knockout F. occidentalis eye-color genes. 1. Wrote manuscript describing results from CRISPR-mediated gene knockout experiments which was published in Insect Molecular Biology (see doi: 10.1111/imb.12913). 2. In addition, we mined the newly published Thrips genome assembly for promoters that will be used in the next phase of F. occidentalis tool development - making transgenic thrips.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Jinlong Han, William Klobasa, Lucas de Oliveira, Dorith Rotenberg, Anna E. Whitfield, Marc� D. Lorenzen. CRISPR/Cas9-mediated genome editing of Frankliniella occidentalis, the western flower thrips, via embryonic microinjection. Insect Mol Biol. 2024 Dec;33(6):589-600. doi: 10.1111/imb.12913. Epub 2024 Apr 27.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Whitfield, A. E. Dissecting the molecular interactions between plant viruses and their arthropod vectors. Southeastern Regional Virology Conference, April 12-14, 2024, Emory University, Atlanta GA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Whitfield, A. E. Plant Viruses as Friends and Foes. Crops Conference. HudsonAlpha Institute for Biotechnology, June 3-6, 2024. Huntsville AL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Whitfield, A. E. Emerging Vector-Borne Plant Diseases at the Intersection of Food Security and Health. Keynote Talk at the 2024 Pandemic Prediction and Prevention Destination Area Symposium. May 14, 2024. Virginia Tech, Blacksburg, VA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Whitfield, A. E. Dissecting the molecular interplay between plant viruses and insect vectors. XX International Plant Protection Congress. Athens, Greece July 1-5, 2024. *Keynote talk for session on Plant Pathogens and Insect vector interactions.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Rotenberg, D. From guts to glands: The journey of a plant virus in an insect vector and lessons learned along the way. 2024 Annual Entomological Society of America, Physiology, Biochemistry and Toxicology Section (Business Meeting), Legacy Research and Impact Talk - PBT Recognition Award, 2024. November 11, 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Rotenberg, D. Advances and innovations in the characterization of molecular interactions between Frankliniella occidentalis and tomato spotted wilt virus. Department of Entomology and Nematology, Seminar Series, University of California, Davis, CA. February 26, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Rotenberg, D. Towards defining molecular determinants of thrips vector competence to transmit tomato spotted wilt virus. Department of Entomology and Plant Pathology, Seminar Series, Auburn University, AL. April 17, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Rotenberg, D. Towards defining molecular determinants of thrips vector competence to transmit tomato spotted wilt virus. Department of Entomology, Seminar Series, Texas A&M University, College Station, TX. November 3, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lorenzen, M.D. Molecular Genetic and Genomic Tools for Peregrinus maidis, an Emerging Model for the Hemiptera. ESA symposium, National Harbor, MD. November 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Lorenzen, M.D. Trials and Tribulations of Making Transgenic Hemipterans. Entomological Society of Queensland, Queensland, Australia. May 2024 [VIRTUAL]


Progress 08/01/22 to 07/31/23

Outputs
Target Audience:Target audiences reached include undergraduate students and the graduate students that participated in the research and received training by the PIs. Research findings were presented to scientists, extension faculty, and stakeholders at local, regional, and national meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Findings from this project were used in lectures and guest lectures in graduate courses (Molecular Plant Microbe Interactions and Vector Transmission of Plant Pathogens at NCSU) to describe current research on vector transmission of plant pathogens and in presentations to scientists and extension faculty. The project has contributed to the training of 2 graduate students, 1 research associate, and an undergraduate researcher. How have the results been disseminated to communities of interest?The research has been presented to scientists, extension faculty, and stakeholders at local, regional, and national meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Develop methods for microinjecting precellular F. occidentalis embryos. 1. Established reliable methods for collecting and microinjecting pre-cellular thrips embryos. 2. Collected and recorded survival data. 3. Wrote detailed protocol which will be submitted for publication shortly. Objective 2. Use the CRISPR/Cas9 system to knockout F. occidentalis eye-color genes. 1. Designed and microinjected guide RNAs targetting eye color genes. 2. Documented results. 3. Wrote manuscript describing results from CRISPR-mediated gene knockout experiments which will be submitted for publication shortly.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Whitfield, Anna E. Never say never: Lessons learned from negative-strand RNA viruses that infect insects and plants. Department of Plant Pathology Seminar Series, Texas A&M University, March 29, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Whitfield, Anna E. Towards defining the viral and vector determinants of tomato spotted wilt virus transmission by thrips. Department of Plant Pathology Seminar Series, The Ohio State University, February 27, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Whitfield, Anna E. Towards defining the viral and vector determinants of tomato spotted wilt virus transmission by thrips. 19e Rencontres Virologie V�g�tale - RVV 2023. Aussois France, January 15-19, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Whitfield, Anna E. Illuminating virus biology in plant and insect hosts: The dedication of the Michael M. Goodin Center for Agricultural Fluorescence Microscopy Experiments and Biological Imaging. August 29, 2022, University of Kentucky, Department of Plant Pathology.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Whitfield, Anna E. What Are Frontiers in Plant Virology After 40 Years of ASV? Plant Virus Interactions with Insect Vectors. 2022. American Society for Virology Meeting, Madison, WI, July 16-20, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Whitfield, Anna E. New strategies for tospovirus and thrips control. 2022 Joint SEB & APS-CD Meeting, San Juan, Puerto Rico, March 26-30, 2022.


Progress 08/01/21 to 07/31/22

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A technician and two students (1 grad and 1 undergrad), as well as a postdoc have been trained in the design and use of the CRISPR/Cas9 gene editing system, as well as microinjection of thrips embryos. 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?We plan to characterize the putative CRISPR edits in the newly established eye-color strains and document their phenotypes. The protocol will be submitted to JoVE and the overall results will be submitted to an appropriate peer-reviewed journal. We will also present results at meetings etc to ensure the results reach the widest possible audience.

Impacts
What was accomplished under these goals? As outlined on the previous page, we developed methods for microinjecting precellular Frankliniella occidentalis embryos and used those methods to test the ability of the CRISPR/Cas9 system to knockout F. occidentalis eye-color genes. Specifically, we performed proof-of-principle experiments to develop a working system for genome-editing in F. occidentalis. We targeted two eye-color genes, white and cinnabar, for ease of visual phenotyping. Cohorts of age-synchronized females were allowed to oviposit into oviposition chambers containing a dilute sucrose solution. Precellular embryos (three-hours-old) were collected and positioned on double-sided strips of tape on a microscope slide in preparation for microinjection. Cas9 protein was complexed with guide RNAs and injected into the precellular embryos using a tapered, quartz needle. Embryos were incubated on agar plates under high humidity at 25C. Once embryos developed a pigmented eye (~48 hours post injection), they were transferred to moistened filter paper to hatch. The hatch rates for white- and cinnabar-targeted embryo cohorts was 24% (25/104) and 30% (16/53), respectively. Hatchlings were then transferred to rearing cups filled with green bean pods and monitored daily for eye-color phenotypes. Of the 25 hatchlings in the white-gene target group, 17 exhibited a faint, yellow eye pigment. Both white and cinnabar eye-color KO males and females (G0 generation) were identified and transferred into rearing cups to establish KO lines. At the time of preparing this report 90 progeny (G1 generation) of the white KO colony exhibited the KO phenotype. PCR amplicons were successfully generated and we are awaiting DNA sequencing results to confirm the presence of CRISPR-mediated insertions/deletions.

Publications