Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 206 - Basic Plant Biology | 999 - Citrus, general/other | 1080 - Genetics (excludes breeding) | 90% |
| 607 - Consumer Economics | 999 - Citrus, general/other | 3010 - Economics | 10% |
Huanglongbing (HLB or citrus greening disease) is an insect-vectored bacterial disease that is decimating the U.S. citrus industry. This is of tremendous concern as citrus is the most economically valuable fruit crop in the US, with revenues from fruit, juice, soft drinks, cosmetics, cleaning products, as well as other processed by-products. This situation is poised to intensify as well; recent studies indicate that crop losses due to damage via insect pests is going to be significantly exacerbated by climate warming, with yield losses estimated to be between 10-25% for each degree Celsius rise in temperature. Development of new approaches and new tools to combat HLB is essential to the survival of the US citrus industry. Functional genomics approaches have not yet been widely utilized to enhance disease resistance, increase stress tolerance, or to modify plant growth or yield in citrus. For example, approaches based on gene-editing techniques like CRISPR/Cas9 are just beginning to be employed in citrus. Recent results from our research team have shown that citrus-specific improvements to CRISPR/Cas9 methodologies can increase the success rate of this approach for inactivating endogenous genes by approximately 20-fold. With these improvements, largescale editing of citrus genes has now become a realistic approach to screen for HLB susceptibility genes. The overall goal of this two-year project is to leverage the extensive molecular information available for HLB infected citrus to develop a large-scale population of gene-edited Valencia plants using multiplex CRISPR/Cas9 technology that can be screened for tolerance to HLB. This project will provide a sustainable resource (i.e. a collection of citrus mutants affecting 1200 genes) for the citrus research community that can also be screened for resistance to other diseases and other value-added traits in the future, and thus accelerate discoveries that would benefit the U.S. citrus industry for many years. The collection of citrus mutants will contribute in achieving many goals of the funding program, including the development of HLB resistance in citrus, the study of how specific citrus genetic pathways contribute to HLB susceptibility, and the identification of candidate genes in citrus that could be intervention targets. As part of this project, we also propose to study the economic and societal impact of using technologies like CRISPR/Cas9 to create new citrus cultivars, with a focus on assessing the acceptance of gene-edited crops by consumers and growers.
Bioinformatics: Develop gene listsMolecular biology: construct Crispr/Cas9 constructs; validation of mutations via PCRPlant tissue culture: transformation into ValenciaFocus group surveys and outreach: to consumers and stakeholdersEvaluation will be through (1) survey returns, (2) advisory meetings, (3) publication in trade journals, (4) publication in peer-reviewed journals, (5) quantitation of amount of access to website and database.
Target Audience
The PIs and postdoctoral associates supported by this award have presented this project in various formats to the scientific and wider general public communities. A list of these efforts in the past year is reported below: Irish, V.F.: Porter Lecture, 'Citrus Development and Disease'. Friday Evening Lecture to the public, Marine Biological Laboratories (and simulcast), August 7, 2023 - Wood Hole, Massachusetts Cintia Sagawa: Talk: Combating bacterial diseases in citrus with genome editing. Plant Molecular Biology Program, MCDB Department, Yale University. April 17, 2023 - New Haven, CT Cintia Sagawa: International Society of Molecular Plant Microbe Interactions (IS-MPMI) Congress. Selected Talk for the Competition "Next Big Idea": Knocking out the yellow dragon disease to save citrus. July 16-20, 2023 - Providence, RI Cintia Sagawa: International Society of Molecular Plant Microbe Interactions (IS-MPMI) Congress. Poster: Identification of HLB susceptibility genes in a Citruspopulation generated using multiplexed CRISPR/Cas9 gene editing. July 16-20, 2023 - Providence, RI Cintia Sagawa, Geoffrey Thomson, Benoit Mermaz: Molecular, Cellular, and Developmental Biology Department Retreat, Yale University. Talk: Combating citrus greening disease with gene editing. September 8-9, 2023 - Woods Hole, MA Cintia Sagawa: Plant Molecular Biology Program, MCDB Department, Yale University. Talk: Strategies to increase gene editing in citrus and combat citrus greening. December 4, 2023 - New Haven, CT Geoffrey Thomson: Talk "Flowering time, Genome engineering, DNA repair" University of Auckland SBS Research Seminar [over Zoom], June 2023 Geoffrey Thomson: Poster: "Gene Editing at Scale in a Perennial Fruit Crop" - ?The International Plant and Animal Genome Conference (PAG31), San Diego CA, January 2024 Benoit Mermaz: Monthly Twitter series to share the progress, findings, and life on the "citrus" team Benoit Mermaz: Podcast from FarmNetz. Benoit participated in a Podcast to talk about his work at Yale, and the gene editing techniques including CRISPR. The soundtrack (in French) has been posted on FarmNetz blog andTwitter(2023) Benoit Mermaz: Yale Postdoctoral Association's Postdoc Spotlight series. Benoit was interviewed to talk about his research and experience at Yale. The resulting video has been advertised on social media (Instagram, and Twitter). Overall, it has reached over 4,000 viewers. (2023)
Changes / Problems
Nothing Reported
Training & Professional Development
Three postdoctoral associates were trained through this project. They received training in communication, leadership and management skills, and interdisciplinarity, and their progress monitored on a weekly basis. In addition, each posdoctoral associate served as a mentor to an undergraduate student, working on their own mentoring and communication skills. One graduate student was trained in research skills and in communication. Six undergraduates were trained through this project; learning basic research techniques and working in a laboratory environment, and presenting their work to the team.
Dissemination Streams
publication in trade journal Citrograph scientific lectures to interested researchers and growers public lectures to the general public Podcasts Twitter Instagram outreach to 8 grade students Project website including data as well as information for the public on HLB, Crispr/Cas9, description of the project, information on societal acceptance of gene edited crops
Next Reporting Steps
Nothing Reported
Target Audience
the PIs and postdoctoral associates supported by this award have presented this project in various formats to the scientific and wider communities. A list of these efforts is below: Jacob, Y: Genome Editing for Food Security & Environmental Security Workshop, Montreal, Canada (2022).Improving gene targeting in plants using T-DNA amplification (Keynote speaker). Irish, V.F.: Invited Speaker, 'Citrus Development and Disease'. Boyce Thompson Institute, Cornell Univ., Ithaca, NY, Aug 2022 Irish V.F.: Invited Speaker, 'Citrus Development and Disease', Botany 2023 Conference, Boise, Idaho, July, 2023 Irish, V.F.: Porter Lecture, 'Citrus Development and Disease'. Friday Evening Lecture to the public, Woods Hole, MA (and simulcast), August 2023 Sagawa, C.: July 13, 2023 - Presented 'Combating disease in citrus' to 8th graders in the Ulysses S. Grant summer program at Yale: https://campuspress.yale.edu/usgrant/ Sagawa, C.: July 16 - 20, 2023 - International Society - Molecular Plant-Pathogen Interactions, Providence, RI - Invited talk for the Top 5 Next Big Idea Competition "Knocking out the yellow dragon disease to save citrus" Sagawa, C.: July 16 - 20, 2023 - International Society - Molecular Plant-Pathogen Interactions, Providence, RI- Poster "Identification of HLB Susceptibility Genes in aCitrus Population Generated Using Multiplexed CRISPR/Cas9 Gene Editing" Mermaz, B.: 2022-present Weekly Twitter update, to share the progress, findings, and outlook from the project Mermaz, B.: April 2023. FarmNetz Podcast. Dr Mermaz participated in a Podcast to talk about his work at Yale, and the gene editing techniques including CRISPR. The soundtrack has been posted on a blog and onTwitter Mermaz, B.: June 2023. Interview on the Yale Postdoctoral Association's Postdoc Spotlight series. Dr. Mermaz was interviewed to talk about his research and experience at Yale. The resulting video has been advertised on social media (Instagram, andTwitter). Overall, it has reached over 4,000 viewers.
Changes / Problems
Nothing Reported
Training & Professional Development
Three postdoctoral associates were trained through this project. They received training in communication, leadership and management skills, and interdisciplinarity, and their progress monitored on a weekly basis. In addition, each posdoctoral associate served as a mentor to an undergraduate student, working on their own mentoring and communication skills. One graduate student was trained in research skills and in communication. Three undergraduates were trained through this project; learning basic research techniques and working in a laboratory environment, and presenting their work to the team.
Dissemination Streams
publication in trade journal Citrograph public lectures to the general public Podcasts Twitter outreach to 8 grade students Project website including data as well as information for the public on HLB, Crispr/Cas9, description of the project, information on societal acceptance of gene edited crops (see crisprcitrus.yale.edu)
Next Reporting Steps
This project has a NCE to February 28, 2024. During the remaining months we will complete the genotyping of the ~4000 Carrizo citrange gene edited lines, and cull those that are redundant or do not show adequate levels of editing, to reach our final population of gene edited lines. Discussions with scientists at the Florida Dept of Agriculture are underway to test disease resistance of these lines under field conditions in future. <br><br>
<br>What was accomplished under these goals? To generate the mutations in 1200 genes, we are employing a multiplex Crispr/Cas9 approach to simultaneously target 4 genes at a time; this means that we need to first generate 300 multiplex Crispr/Cas9 constructs, validate them, and introduce them into Agrobacterium for plant transformation. We validated 149/300 planned constructs based on extant Carrizo citrange sequence information; the generation of a refernce quality phased Carrizo citrange genome is a goal of a currently submitted project. In the past year, we have carried out transformations of Carrizo citrange, with the 149/300 constructs now introduced into this cultivar, resulting in over 4000 GFP positive plants that are now undergoing genotyping via sequencing for gene editing events at each of the four target sites per plant. We have also carried out experiments to identify the most efficient way of inducing gene editing in citrus by comparing efficiencies of different promoters (e.g., YAO, 2X35S, RPS5, and UBQ10) driving Cas9 and different promoters (RPS8 and U6) driving the sgRNA cassette. In addition, we have compared two different multiplex CRISPR systems: tRNA-based sgRNA arrays under the control of a single promoter, or expression of each individual sgRNA by its own promoter. We have generated a number of comparable constructs with consistent backbones and sgRNA arrays included to assess these different comparisons and have defined the parameters for efficient multiplex CRISPR/Cas9 gene editing in citrus. Based on these experiments, we settled on using RPS5 to drive Cas9, RPS8 to drive the sgRNA cassette, and the tRNA-based arrays for multiplexing. We have established a website with database entry and searchability to access the sequences, vectors, and plant materials generated by this project, as well as information for consumers and growers (crisprcitrus.yale.edu). We also carried out focus group analyses about consumer purchasing and consumption habits, attitudes towards genetically modified (GM) crops, and customer preferences for different product attributes. Based on these data, we administered an online survey that was administered to 2000 US orange juice customers in 2022, with resulting data analyzed using a mixed logit model. The results indicate that 1) CRISPR products have the potential to perform better in the marketplace as compared to 'GM' products, 2) ethical concerns may be an issue, and 3) educational campaigns could make a difference, especially with specific target groups. In addition, we have been active in presenting the results of our work to other researchers and to the public through talks, publications, web-based outreach and social media (e.g., project website). <br><br><b>Publications</b><br>
Target Audience
Citrus researchers interested in using Crispr approaches; citrus researcher, citrus growers, and other stakeholders interested in solutions to HLB; federal, state and local regulatory personnel involved in citrus production; consumers.
Changes / Problems
Nothing Reported
Training & Professional Development
Meetings of the entire team occur bimonthly (via zoom) to discuss progress, emerging issues, and to discuss how best to present information to the public and stakeholders. Meetings of the Yale team occur weekly, with updates to progress on the cloning, transformation, and characterization of engineered citrus plants. Through this project, we have provided training and professional development opportunities for one University of Florida graduate student in agricultural economics, one postgraduate student at Yale University in plant tissue culture and molecular biology, and three Yale university postdoctoral associates in plant tissue culture, molecular biology, project design, and communication. Restrictions due to the Covid-19 pandemic have limited our ability to carry outreach activities in person, but we aim to ramp up these activities in the coming year.
Dissemination Streams
A website, https://crisprcitrus.org/, has been posted to direct interested parties to the planned and completed work of the project.
Next Reporting Steps
• Based on the results from our current experiments to define the optimum construct type for citrus transformation (as described above), we will generate the remaining 151 constructs, and introduce these into Carrizo citrange. We will establish a testing regime to prescreen lines for further testing for HLB tolerance/resistance. • We will continue to test out protocols for Valencia transformation. • We will complete the large scale survey of consumer acceptance and fully analyze the data. • We will assemble our data into one or more manuscripts to report these findings. <br><br>
<br>What was accomplished under these goals? To generate the mutations in 1200 genes, we are employing a multiplex Crispr/Cas9 approach to simultaneously mutate 4 genes at a time; this means that we need to first generate 300 multiplex Crispr/Cas9 constructs, validate them, and introduce them into Agrobacterium for plant transformation. The constructs each contain 35Sp-GFP-nptII (for selection), YAOp-CAS9 and 4 distinct independently synthesized sgRNA sequences each driven by the U6 promoter; these are being assembled using MoClo cloning. As of August, 2021, we had 149/300 constructs cloned, sequenced, and transformed into Agrobacterium. In the past year, using these constructs, we have obtained 77 GFP positive Valencia plants that are now in soil. In addition, we have carried out transformations of these constructs into Carrizo citrange, with 198 GFP positive Carrizo citrange plants now in hand. These plants were validated as GFP positive based both on expression of the GFP marker and PCR assessment of integration of the construct into the genome. We are currently in the process of genotyping these plants via sequence analyses for gene editing events at each of the four target sites. These sequence data are being analyzed using the Inference of Crispr Edits tool to estimate the indels at each site. We have also intitiated experiments to identify the most efficient way of inducing gene editing in citrus, comparing efficiencies of different promoters (YAO, 2X35S, and UBQ10) driving Cas9; different promoters (RPS8; U6) driving the sgRNA cassette, and comparisons of tRNA-based arrays versus individually expressed sgRNA. We have generated a number of comparison constructs with consistent backbones and arrays included to assess these different comparisons. These comparisons are currently being transformed into plants and will provide more information on best approaches to synthesize the remaining 151 planned constructs. Based on suggestions that came out of the 2022 ECDRE Project Director's meeting, we will focus on generating the completed population in Carrizo citrange. Because of this, we will need to redisign some constructs in terms of specific sgRNAsequences so as to work effectively in Carrizo citrange. We have established a website with database entry and searchability to access the materials generated by this project. Based on the prior results we obtained from focus groups about consumer purchasing and consumption habits related to fresh fruits and vegetables (particularly citrus), their attitudes towards biotechnology, and their preferences for different product attributes, we have developed an online survey that was administered to 2000 US orange juice customers in 2022. A soft launch was carried out and the initial data obtained is currently being assessed using a mixed logit model. The preliminary results indicate that 'CRISPR' products have the potnetial to perform better in the marketplace as compared to 'GM' products; ethical concerns may be an issue; risk-loving individuals are more accepting of CRISPR, and that educational campaigns could make a difference, especially with specific target groups. <br><br><b>Publications</b><br>
Target Audience
Nothing Reported
Changes / Problems
Nothing Reported
Training & Professional Development
Nothing Reported
Dissemination Streams
A website, https://crisprcitrus.org/, has been posted to direct interested parties to the planned work of the project.
Next Reporting Steps
We will generate the remaining 151 constructs, and introduce them into Valencia. We will continue to test out protocols for Valencia transformation. We anticipate generating a population of 1200 Valencia plants for further testing; we will define suitable testing locations and regimes for this work. We will carry out focus group meetings. We will carry out a large scale survey of consumer acceptance. We will assemble our data into one or more manuscripts to report these findings. <br><br>
<br>What was accomplished under these goals? We commenced the project on January 1, 2021. To generate the mutations in 1200 genes, we are employing a multiplex Crispr/Cas9 approach to simultaneously mutate 4 genes at a time; this means that we need to first generate 300 multiplex Crispr/Cas9 constructs, validate them, and introduce them into Agrobacterium for plant transformation. The constructs each contain 35Sp-GFP-nptII (for selection), YAOp-CAS9 and 4 distinct independently synthesized sgRNA sequences each driven by the U6 promoter; these are being assembled using MoClo cloning. As of August, 2021, we have 149/300 constructs cloned, sequenced, and transformed into Agrobacterium. Using these constructs, we have carried out 196 transformations into Valencia orange epicotyl segments. Of these, so far we have obtained 36 positive Valencia plant shoots from 16 constructs. In order to optimize this pipeline, we are testing out 6 different protocols for Valencia transformation. Our goal is to have three plant replicates for each constuct, totaling 1200 plants. We have established a website with database entry and searchability to access the materials generated by this project. To assess societal impact, we designed a first draft of the focus group script, which we will use to gather initial information about consumer purchasing and consumption habits related to fresh fruits and vegetables (particularly citrus), their attitudes towards biotechnology, and their preferences for different product attributes. We plan to finalize the focus group script and start recruiting subjects to participate in the focus group meetings by September 2021.Secondly, we have a rough draft of the survey questionnaire ready. This survey will be used to collect more detailed information from a representative sample of consumers. The survey questionnaire is being designed on the Qualtrics platform, and it contains questions related to citrus consumption, CRISPR valuation, attitude and beliefs towards biotechnology, and other behavioral traits (such as risk perceptions and preferences, ambiguity tolerance etc.). The data gathered from the focus group will be used to refine some of the details in this survey. We plan to finalize the survey and field it for data collection shortly after completion of the focus group meetings (estimated timeline of October/November 2021). <br><br><b>Publications</b><br>