Innovation in soybean trait development is dependent on foundational research and tools that will allow for discovery, validation, and the ability to efficiently deploy traits into elite germplasm. The aim of this program is to create open access tools that will empower soybean scientists conducting research across all roadmaps of the LRSP by accelerating discovery, enabling precision editing, and shortening the time needed to breed newly discovered traits into high yielding cultivars. These tools include both software and lab components in order to create a gene editing research and development pipeline for the soybean research community that carries through from discovery to pre-commercialization.

CRISPR, which stands for "clustered regularly interspaced short palindromic repeats” is a powerful new molecular biology tool for gene editing and crop improvement. It has been gaining attention and is predicted to be one of the most impactful discoveries in science for the next decade. Current editing approaches rely almost exclusively on old transformation methodology to introduce CRISPR components, and almost all approaches are taking a ‘deletion’ approach, or making short edits, rather than inserting larger stretches of DNA for more ambitious projects potentially leading to higher impact products for the farmer and consumer. There would be benefit across the soybean research community in addressing these issues. Additionally, the initial CRISPR discovery revolved around an enzyme called CAS-9: new research has uncovered several other enzymes that do the same job more efficiently. There may be an opportunity to utilize one of these enzymes or to develop a suite of enzymes especially suited for soybean. In doing so, a pathway may be created that affords freedom to operate in both research and product development spaces.

1. Create WCIC website Soy Portal by February 2019.
2. Deposit soy-optimized Golden Gate components by September 2019.
3. List new soy cultivars as available for service at WCIC on website by September 2020.
4. Make public report of non-Agro transformation process by September 2021.
5. Make public report of editing efficiency across enzymes and genes by September 2021.
6. Make public report of advances on nanoparticle-based, non-transgenic editing efficiency by September 2021.

Updated May 5, 2020:
Research Aims for this 3-year project are to:
1. Create a Soy portal on the WCIC website
2. Create optimized soybean editing vector components using Golden Gate cloning
3. Optimize meristem-based transformation/editing protocols for new soybean cultivars
4. Improve efficiency of non-Agrobacterium, meristem-based transformation systems for soybean
5. Compare efficiency of gene editing emzymes
6. Assess gene editing efficiency across multiple gene targets
7. Investigate nanoparticle-mediated editing in soybean

Funding for the proposed research was received in October and project work initiated. During Quarter #1 of this project (Oct. 1-Dec. 15), we initially proposed to begin research/steps to address Aims 1, 2, and 7.

For Aim 1, we have begun work on modifying the WCIC website to include additional information on soybean transformation/editing services and options, and have updated the list of current varieties/elite lines that can be selected for transformation.

Further work on Aim 2 will continue into Quarter #2 and will include adding information on soybean transformation protocol steps, adding links to external resources for researchers considering soybean transformation/editing projects, and setting up a soybean-specific portal for researchers to more easily access service features. Work initiated under Aim 2 in Quarter #1 included design of optimized soybean transformation and editing vectors, and adapting sequences of soybean optimal components (e.g. promoters, Cas9 gene, selectable/screenable marker genes, etc.) for Golden Gate cloning for vector assembly. Component and vector assembly and testing will continue into Quarter #2.

Research work focused on Aim 7 included aligning collaborators in biomaterials/nanoparticle-based editing research for design and construction of nanoparticles containing editing reagents (Cas9 protein and guide RNAs). Target tissues for the research in soybean were discussed and selected, and generation of target material is underway. Research in the area of Aim 7 will continue into Quarter #2.

Finally, while research in the area of Aim 3 was not initially proposed to begin until Quarter#3, initial optimization experiments were undertaken for testing meristem-based transformation of soybean to enhance efficiency of the process and test targeting of alternative genotypes. Research work in the area of Aim 3 will also continue into Quarter #2.

Updated May 5, 2020:
Research Aims for this 3-year project are to:
1. Create a Soy portal on the WCIC website
2. Create optimized soybean editing vector components using Golden Gate cloning
3. Optimize meristem-based transformation/editing protocols for new soybean cultivars
4. Improve efficiency of non-Agrobacterium, meristem-based transformation systems for soybean
5. Compare efficiency of gene editing enzymes
6. Assess gene editing efficiency across multiple gene targets
7. Investigate nanoparticle-mediated editing in soybean

During Quarter #2 of this project (Dec. 15, 2018-Mar. 15, 2019), we proposed to continue research/steps addressing Aims 1, 2, and 7, and also initiated research for Aim 3.

For Aim 1, we have continued to work on modifying the WCIC website to include additional information on soybean transformation/editing services and options, and have updated the list of current varieties/elite lines that can be selected for transformation.

Work on Aim 2 during Q2 included construction work on constructs containing 12 guideRNAs targeting editing marker genes BIGSEED and GLABROUS for editing confirmation research. We are working on two types constructs, in one, the guide RNA cassette is driven by a Pol III promoter (GmU6-16g-1) versus in the other construct which will use a Pol II promoter. This will be tested in the case that the Pol III promoter is not effectively driving the long, polycistronic guide RNA cassette then the guides near the end (the ones targeting GLABROUS) will be present in reduced copy number, thus one might expect to see bald plants at a lower frequency than when a strong Pol II promoter yields full length transcripts. Vector construction work during Q2 also included design of optimized soybean transformation and editing vectors, and adapting sequences of soybean optimal components (e.g. promoters, Cas9 gene, selectable/screenable marker genes, etc.) for Golden Gate cloning for vector assembly. Component and vector assembly and testing will continue into Q3.

Research directed at Aim 3 during Q2 included testing of different Agrobacterium strains and treatment protocols in a meristem-based transformation system targeting soybean, selection/regeneration parameter optimization, and development and optimization of enhanced explants (targeted for transformation) that can be mechanically isolated, dehydrated, stored, rehydrated and remain viable and responsive/competent to transformation/editing. Current soybean genotypes targeted for meristem-based transformation include the following: Williams82, Williams82- ssd (Stupar), 3025N, 3613N, 3849N, LD10-30087, LD10-30092, LD10-30094, LD10-30080, LD10-30084, LD10-30110.

Research work focused on Aim 7 included initiation of investigations aimed at RNP nanoparticle design and construction, and initial delivery/confirmation of nanoparticle uptake into plant cell cultures and tissues. Preliminary experiments testing nanoparticle-mediated gene editing are planned for Q3.

Updated May 5, 2020:
Research Aims for this 3-year project are to:
1. Create a Soy portal on the WCIC website
2. Create optimized soybean editing vector components using Golden Gate cloning
3. Optimize meristem-based transformation/editing protocols for new soybean cultivars
4. Improve efficiency of non-Agrobacterium, meristem-based transformation systems for soybean
5. Compare efficiency of gene editing enzymes
6. Assess gene editing efficiency across multiple gene targets
7. Investigate nanoparticle-mediated editing in soybean

During Quarter #3 of this project (Mar. 16-June 15, 2019), we continued research/steps addressing Aims 1, 2, and 7, and also initiated research for Aim 3.

For Aim 1, we have continued to work on modifying the WCIC website to include additional information on soybean transformation/editing services and options, and continue to updated the list of current varieties/elite lines that can be selected for transformation.

Work on Aim 2 during Q3 continued with construction work on plasmids containing multiple guideRNAs targeting editing marker genes BIGSEED and GLABROUS for editing confirmation research. We are working on two types constructs, in one, the guide RNA cassette is driven by a Pol III promoter (GmU6-16g-1) versus in the other construct which will use a Pol II promoter. This will be tested in the case that the Pol III promoter is not effectively driving the long, polycistronic guide RNA cassette then the guides near the end (the ones targeting GLABROUS) will be present in reduced copy number, thus one might expect to see bald plants at a lower frequency than when a strong Pol II promoter yields full length transcripts. Vector construction work during Q3 also included continued design of optimized soybean transformation and editing vectors, and adapting sequences of soybean optimal components (e.g. promoters, Cas9 gene, selectable/screenable marker genes, etc.) for Golden Gate cloning for vector assembly. Component and vector assembly and testing will continue into Q4.

Research directed at Aim 3 during Q3 included testing of different Agrobacterium strains and treatment protocols in a meristem-based transformation system targeting several different genotypes of soybean, selection/regeneration parameter optimization, and development and optimization of enhanced explants (targeted for transformation) that can be mechanically isolated, dehydrated, stored, rehydrated and remain viable and responsive/competent to transformation/editing. Current soybean genotypes targeted for meristem-based transformation include the following: Williams82, Williams82- ssd (Stupar), 3025N, 3613N, 3849N, LD10-30087, LD10-30092, LD10-30094, LD10-30080, LD10-30084, LD10-30110.

Research work focused on Aim 7 included initiation of further investigations aimed at RNP nanoparticle design and construction, and initial delivery/confirmation of nanoparticle uptake into plant cell cultures and tissues. In preliminary replicated experiments testing nanoparticle-mediated DNA delivery into soybean embryonic axis explants, transient expression of the screenable marker gene, GUS, was documented in desired meristem regions, confirming DNA delivery into and expression within target cells. Additional experiments aimed at optimization of nanoparticle and DNA delivery parameters are planned for Q4 with the goal of achieving stable transformation and transmission to T1 progeny. Invention disclosure to WARF is planned when L2 stable transformation is determined.

Updated January 12, 2021:
Research Aims for this 3-year project are to:
1. Create a Soy portal on the WCIC website
2. Create optimized soybean editing vector components using Golden Gate cloning
3. Optimize meristem-based transformation/editing protocols for new soybean cultivars
4. Improve efficiency of non-Agrobacterium, meristem-based transformation systems for soybean
5. Compare efficiency of gene editing enzymes
6. Assess gene editing efficiency across multiple gene targets
7. Investigate nanoparticle-mediated editing in soybean
Funding for the proposed research was received in October (2018) and project work initiated at that time. During Quarter#4 of this project (June 16-September 15, 2019), we continued research/steps addressing Aims 1, 2, 3, 4 and 7. For Aim 1, we have continued to work on modifying the WCIC website to include additional information on soybean transformation/editing services and options, and continue to interact with the soybean genomics community to determine optimal target genotypes and to add those to the list of genotypes available through the transformation service pipeline. As new genotypes become a target option, they will be added to the website list for customer orders. Current soybean genotypes available for public transformation services include Williams82, IL3025N, and IL3613N. Work on Aim 2 during Q4 continued with construction work on plasmids containing multiple guideRNAs ( up to 12) targeting editing marker genes BIGSEED and GLABROUS for editing confirmation research. We are working on two types constructs, in one, the guide RNA cassette is driven by a Pol III promoter (GmU6-16g-1) versus in the other construct which uses a Pol II promoter. The Pol II promoter is being tested in the case that the Pol III promoter is not effective in driving the long, polycistronic guide RNA cassette. If that is the case (when testing the Pol III promoter), then the guides near the end (the ones targeting GLABROUS) will be present in reduced copy number, thus one might expect to see bald plants at a lower frequency than when a strong Pol II promoter yields full length transcripts. Vector construction work during Q4 also included continued design of optimized soybean transformation and editing base vectors, and adapting sequences of soybean optimal components (eg.promoters, Cas9 gene, selectable/screenable marker genes, etc.) for Golden Gate cloning for vector assembly. Component and vector assembly and testing will continue into Q4. Research directed at Aim3 during Q4 included experiments in which higher soybean transformation frequency was achieved by modifying culture transfer steps. The protocol changes are now currently being evaluated in large scale transformation service production pipeline experiments. Studies were also initiated in Q4 to investigate the effect of pre-culture conditions on soybean transformation efficiency. Modifications to the methods used to produce soybean embryonic axis explants (the starting material for meristem transformations) are underway to optimize explant isolation efficiency and viability. During Q4, as in previous research quarters, new soybean genotypes have been tested in our “standard” meristem-based transformation system to determine success and frequency of transformation. To date, the following genotypes have been successfully (germline) transformed through this system (at varying efficiencies): Williams82, single seed decent Williams 82 (Stupar), IL3025N, IL3613N, IL3849N, IL2643N, LD10-30087, LD10-30092, LD10-30094, LD10-30080, LD10-30084, LD10-30110, MN0810CN, Hikmok, MN1312CN, M09-876048, MN1806CN. Research on Aim 4 was initiated through initial probe experiments to study the potential of micro-fiber mediated meristem transformation. Transient expression of transgenes was observed at a low frequency following preliminary experiments. Further research is underway to optimized micro-particle based DNA delivery parameters. In addition to the micro-fiber based DNA delivery research, we have continued to optimize biolistic (gene-gun) based transformation of soybean in our meristem transformation systems. In Q4 we have routinely transformed various soybean genotypes via gene gun mediated DNA delivery and now offer this as a transformation service option to customers. Finally, in Q4, research work focused on Aim 7 included further experiments aimed at optimizing transgene cassette and RNP nanoparticle design, construction, and initial delivery/confirmation of nanoparticle uptake into plant cell cultures and tissues. Marker dye and DNA delivery into plant cell cultures/tissues was tested using two new nanoparticle types/sizes. Positive expression of the visual marker gene encoding green fluorescent protein (GFP) was observed in specific nanoparticle delivery treatments, demonstrating delivery of functional expression cassettes into somatic cells. Further experiments are underway to replicate results, and optimize nanoparticle characteristics and DNA/RNA/protein content for transgenic and editing applications in plant cell culture and regeneration systems, with the goal of germline transformation /editing.

Updated December 2, 2021:
The overall goal of this research project was to conduct research to develop and refine soybean genetic engineering and gene editing systems that are efficient, robust, publicly available, and genotype-flexible for use in soybean functional genomics research and genetic improvement/breeding efforts. The impact of reaching the goals would aid in reducing the time and costs of soybean engineering/editing, enable transformation/editing of any preferred cultivar, and improve the accessibility and speed of genetic study and improvement of soybean in both the public and private sectors. Specific aims of this project included:
1. Creation of an updated and user-friendly Soybean Transformation/Editing portal on the website of the Wisconsin Crop Innovation Center transformation/editing service facility.
2. Construction and deployment of optimized soybean engineering/editing vectors.
3. Optimization of meristem-based transformation/editing protocols for multiple soybean cultivars.
4. Improvement in efficiency of non-Agrobacterium, meristem-based transformation systems for soybean.
5. Comparison of efficiency of various gene editing enzymes and systems.
6. Assessment of transformation and gene editing efficiency across multiple gene targets in soybean.
7. Investigation of nanoparticle-mediated editing/engineering systems for use in soybean that would enable transgene-free germline editing.

New technology and traits will be able to advance more rapidly and efficiently