Source: UNIVERSITY OF FLORIDA submitted to
COMPARISON OF ON AND OFF-TARGET EFFECTS OF EPIGENETIC EDITING, TARGETED MUTAGENESIS AND RNAI FOR SUPPRESSION OF FLOWERING IN ENERGYCANE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1030914
Grant No.
2023-33522-40411
Cumulative Award Amt.
$650,000.00
Proposal No.
2023-02448
Multistate No.
(N/A)
Project Start Date
Aug 1, 2023
Project End Date
Jul 31, 2027
Grant Year
2023
Program Code
[HX]- Biotechnology Risk Assessment
Project Director
Altpeter, F.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
U.S. DOE identified energycane (Saccharum spp hybrid) as ideal biofuel crop for the Southeast. This project will compare on and off-target effects of alternative biotechnology approaches for complete suppression of flowering in energycane. This will elevate biocontainment by preventing gene flow to sexually compatible weedy relatives and inform regulators about the precision and effectiveness of the alternative approaches.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012020104030%
2012020105010%
2012020108030%
2012020108130%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2020 - Sugar cane;

Field Of Science
1040 - Molecular biology; 1081 - Breeding; 1080 - Genetics; 1050 - Developmental biology;
Goals / Objectives
Long-term goal: To create elite energycane cultivars with complete and consistent flowering suppression. Selection of the superior approach for suppression of flowering will be informed by comparing on and off-target effects of the different alternative approaches alongside agronomic performance.RNAi has recently confirmed the target florigens for complete suppression of flowering, contributing to elevated biosafety and biomass production in energycane in the PI's research program. However, RNAi approaches require continuous expression of transgenes to obtain the desired phenotype, which may vary depending on environmental conditions and transgene integration structure. Objectives of this proposal are to compare flowering suppression with CRISPR mediated mutagenesis and epigenetic editing of the confirmed florigens to provide flowering suppression without requiring continuous transgene expression. To refine this approach, gene and epigenetic editing cassettes will be removed from a subset of antibiotic resistant and regenerating calli via inducible expression of Cre-lox site specific recombination. This will allow to explore the stability of the editing in the absence of the editing cassettes and reduce potential off-target effects of the gene and epigenetic editing tools.
Project Methods
1. To develop elite energycane lines with suppression of confirmed target FT genes to promote (trans)gene containment using biolistic transfer of gene editing or epigenetic editing constructs.2. Evaluate off-target effects of gene editing, epigenome editing and Cre-lox recombination at the genotype level using target enrichment sequencing of most likely off-target regions.3. Compare the flowering suppression and agronomic performance of elite energycane lines using gene editing, epigenome editing or RNAi in a two-year field trial (plant cane and ratoon).

Progress 08/01/23 to 07/31/24

Outputs
Target Audience:During the reporting period the project personnel gave 11presentations on gene editing and genetic modification of energycane and sugarcane at international conferences, research institutions and industry. The target audience of this project reached during this reporting period include: Academic researchers: Scientists and scholars at universities and research institutions who are studying potential risks associated with genetically engineered crops, including bioenergy feedstock crops like energycane. Government scientists: Researchers working in federal or state agencies who are involved in assessing the safety and environmental impacts of biotechnology. Agricultural scientists: Experts focused on understanding how genetically engineered crops might impact agricultural ecosystems and practices. Environmental scientists: Researchers studying the potential effects of biotechnology on natural ecosystems and biodiversity. Policy makers: Individuals involved in developing regulations and policies related to biotechnology and genetically modified organisms. Industry researchers: Scientists working in biotechnology companies who are interested in understanding and mitigating potential risks associated with their products. Changes/Problems:Construction of the different recombinant DNA vectors for the various editing approaches was more challenging than anticipated and caused some delays. All required recombinant DNA vectors have now been completed and were introduced into energycane. What opportunities for training and professional development has the project provided?The project provided support for 2 research scholars from Vietnam and Argentina, respectively and 1 biological scientist who trained the research scholars in tissue culture,vector construction and genetic transformation. How have the results been disseminated to communities of interest?As presentations at conferences, research institutions and industry. What do you plan to do during the next reporting period to accomplish the goals?Additional independent transgenic lines/plants from the different recombinant DNA constructs will be regenarated and evaluated for the targeted mutations by capillary electrophoresis and sequencing of the PCR amplicons of the target genes or for epigenetic editing by bisulfite sequencing of the target amplicons. The subset of events that was generated with editing constructs that allow removal of the gene editing cassette via inducible expression of cre-lox mediated site specific recombination will be evaluated for the successful removal of the gene editing cassette.

Impacts
What was accomplished under these goals? Recombinant DNA constrcuts were generated, which are expected to direct flowering suppression with CRISPR mediated mutagenesis orepigenetic editing of the confirmed florigens to provide flowering suppression without requiring continuous transgene expression. To refine this approach, additional gene and epigenetic editing constructs were also generated that allow transgene removal via inducible expression of Cre-lox site specific recombination. The different recombinant DNA constructs were introduced into embrypogenic energycane callus. Transgenic plants were regenerated and their transgenic nature was confirmed by PCR for some of the constructs, while others are still undergoing selection on antibiotic containing culture media and others are regenerating plants following selection and will be analyzed to confirm their transgenic nature.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Altpeter, F. The More the Merrier? Editing the Highly Polyploid Sugarcane Genome. World Congress of In Vitro Biology, St. Louis MO June 2024
  • Type: Other Status: Published Year Published: 2024 Citation: Altpeter, F. Editing the Complex Sugarcane Genome. Artia Global Partners, virtual. February 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Maitra, S., Cheng, M.-S., Liu, H., Cao, D.V., Kannan, B., Long, S.P., Shanklin, J., Altpeter, F. and Singh. V. Transgenic energycane as an alternative feedstock for biodiesel production: An industrially relevant study. AIChE Annual Meeting, Orlando FL. November 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Kannan, B., Cano-Alfanar, S., Jamil, A. and Altpeter, F. Field evaluation of transgenic sugarcane with RNAi suppression of FLOWERING LOCUS T (FT) and herbicide resistance. Plant Biology Meeting August 2023, Savannah, GA. P-800-18. 2023
  • Type: Other Status: Published Year Published: 2023 Citation: Altpeter, F. Embracing the Challenge: Editing the Complex Sugarcane Genome, Agricultural Genetic Engineering Research Institute, Cairo, Egypt. August 2023.
  • Type: Other Status: Published Year Published: 2023 Citation: Altpeter, F. Metabolic Engineering and Gene Editing of Bioenergy and Forage Grasses to Combat Climate Change and Fuel the Emerging Bioeconomy, King Abdullah University of Science and Technology, Saudi Arabia. August 2023
  • Type: Other Status: Published Year Published: 2023 Citation: Altpeter, F. Growth Comes from Risk - Accelerating Crop Improvement with Novel Breeding Technologies, Ahmedabad University, Ahmedabad, India. August 2023.
  • Type: Other Status: Published Year Published: 2023 Citation: Altpeter, F. Fueling the Bioeconomy and Combatting Climate Change: Metabolic Engineering and Gene Editing of Forage and Bioenergy Grasses, Ahmedabad University, Ahmedabad, India. August 2023.
  • Type: Other Status: Published Year Published: 2023 Citation: Altpeter, F. Embracing the Challenge: Editing the Highly Polyploid Sugarcane Genome, ICAR, Sugarcane Breeding Institute, Coimbatore, India. August 2023
  • Type: Other Status: Published Year Published: 2023 Citation: Altpeter, F. Editing the Most Complex Crop Genome, Gujarat University, Ahmedabad, India. August 2023.
  • Type: Other Status: Published Year Published: 2023 Citation: Altpeter, F. Towards Oilcane: Fueling the Bioeconomy with Metabolic Engineering and Gene Editing of Sugarcane, National Academy of Agricultural Science, New Delhi, India. August 2023.