Project Details:

Title:
Non-Transgenic Generation of Herbicide Resistance in Soybean Using CRISPR Base Editing

Parent Project: This is the first year of this project.
Checkoff Organization:North Central Soybean Research Program
Categories:Weed control, Breeding & genetics
Organization Project Code:NCSRP
Project Year:2019
Lead Principal Investigator:Feng Qu (Ohio Agricultural Research and Development Center)
Co-Principal Investigators:
Keywords: Trait modification, herbicide tolerance, weed management

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

This is a collaborative project among researchers from four different states (Ohio – Qu and Doohan, Nebraska - Clemente, Missouri - Stacy, and Minnesota – Stupar). This project strives to address the urgent concern that weeds are becoming an increasingly serious threat to soybean production, as existing and emerging weeds become tolerant to the limited selection of herbicides used in soybean fields. We propose to address this problem by equipping soybean with new genetic traits that confer tolerance to three novel classes of herbicides, allowing these herbicides to be used on soybean. Specifically, our project will address two inter-connected goals: (i) establish a CRISPR base editing system that generates herbicide-resistant soybean quickly and non-transgenically; and then (ii) use the new system to produce novel herbicide resistance traits, ideally double or triple herbicide resistance. Upon successful completion of this project, the improved soybean cultivars will be amenable to a wider selection of herbicides, permitting more effective weed control. Importantly, we will develop these new herbicide tolerance traits by primarily using the new CRISPR base editing technology, thus bypassing the strict regulation rules for transgenic crops, allowing soybean farmers faster access to the new soybean varieties.

Project Objectives

• Accelerate the process of CRISPR base editing in soybean by using germinating soybean seed;
• Streamline the CRISPR base editing protocol in multiple soybean cultivars by equipping them with the base editing enzyme BE3;
• Generate three novel herbicide tolerance traits in soybean using the new CRISPR base editing approach.

Project Deliverables

Should the project progress as planned, we expect to garner the following deliverables that will directly benefit soybean growers by increasing the profit of growing soybean while reducing inputs:
• A fast, cost-effective, and non-transgenic base editing protocol for accurately modifying soybean genes without disrupting their functions;
• Multiple soybean lines resistant to diverse herbicides generated with the new base editing technology;
• Multiple soybean cultivars equipped with the base editing BE3 enzyme, as well as other base-editing Cas9 with more advanced characteristics, ready to be utilized by the soybean research community for editing other soybean gene in order to improve soybean seed quality and yield.

Progress of Work

Updated October 1, 2019:
Project title: Non-transgenic generation of herbicide resistance in soybean using CRISPR base editing

This is a collaborative project among researchers from four different states (Ohio – Qu and Doohan, Nebraska - Clemente, Missouri - Stacy, and Minnesota – Stupar), with the goals to (i) establish a base editing system that generates herbicide-resistant soybean quickly and non-transgenically; and then (ii) use the new system to produce novel herbicide resistance traits. Specifically, we proposed to pursue three specific objectives:
• Accelerate base editing in soybean by using germinating soybean seed;
• Streamline the base editing protocol in multiple soybean cultivars by equipping them with the base editing enzyme BE3;
• Generate novel herbicide resistance traits in soybean using the new base editing approach.

Should the project progresses as planned, it is expected to yield the following deliverables that will directly benefit soybean growers by increasing the profit of growing soybean while reducing inputs:
• A fast, cost-effective, and non-transgenic base editing protocol for accurately modifying soybean genes without disrupting their functions;
• Multiple soybean lines resistant to diverse herbicides generated with the new base editing technology;
• Multiple soybean cultivars equipped with the base editing BE3 gene, ready to be utilized by the soybean research community for editing other soybean gene in order to improve soybean seed quality and yield.

In the proposal we set the following milestones and performance indicators for the first year (October 1, 2018 to September 30, 2019):
(1) Successful base editing of the soybean ALS gene by using germinating soybean seed and particle bombardment DNA delivery, generating Imazapyr-resistant soybean seed;
(2) Successful assembly of DNA constructs for producing BE3 (base editor, third generation)-transgenic soybean;
(3) Initiation of soybean transformation to generate BE3-transgenic soybean in a number of soybean cultivars: Williams 82, Thorne, Maverick, Bert, and Jack.

Progress, accomplishments and deliverables to-date:
By now we have carried out six months of NCSRP-funded research. We are making steady progresses towards meeting all three of the milestones.
• Specifically, we have successfully assembled a construct that contains both the BE3 base-editing Cas9 enzyme, and a guide RNA designed to guide BE3 to a specific position in the soybean ALS gene to mediate the editing of specific bases, leading to herbicide (Imazapyr) resistance. This plasmid is named as pWI-BE3-gGmALS. We also produced another construct that would express the BE3 enzyme only, designated as pWI-BE3 (milestone 2).
• We then used both constructs to transform embryogenic tissues generated from young soybean seed, using a particle bombardment procedure. We obtained five (5) transgenic events for pWI-BE3-gGmALS, and one (1) event for pWI-BE3, from which transgenic seedlings are being induced. At this point at least one event appears to contain the successfully base-edited ALS gene. Other events may also yield base-edited seedlings because the construct continues to carry out the editing while the tissues are being induced for seedling differentiation (milestone 1).
• We will next send both the pWI-BE3-gGmALS and pWI-BE3 constructs to collaborators in Nebraska, Missouri, and Minnesota for producing base-edited plants in other soybean varieties (milestone 3).

View uploaded report Word file

Updated September 30, 2019:

Updated October 1, 2019:
First of all, we wish to thank NCSRP for the decision to continued support!
By now we have completed the first year of this NCSRP-funded project. We are proud to announce that we have met the milestones – with the generated soybean plants being reared for seed production.
• Specifically, we have successfully assembled a construct that contains both the BE3 base-editing Cas9 enzyme, and a guide RNA designed to guide BE3 to a specific position in the soybean ALS gene to mediate the editing of specific bases, leading to herbicide (Imazapyr) resistance. This plasmid is named as pWI-BE3-gGmALS. We also produced another construct that would express the BE3 enzyme only, designated as pWI-BE3 (milestone 2).
• Both constructs have been used to transform embryogenic tissues generated from young soybean seed, using a particle bombardment procedure. We obtained five (5) transgenic events for pWI-BE3-gGmALS, and one (1) event for pWI-BE3, from which transgenic seedlings are being induced. At least one event appears to contain the successfully base-edited ALS gene. Other events may also yield base-edited seedlings because the construct continues to carry out the editing while the tissues are being induced for seedling differentiation (milestone 1).
• Both the pWI-BE3-gGmALS and pWI-BE3 constructs have been sent to collaborators in Nebraska, Missouri, and Minnesota for producing base-edited plants in other soybean varieties (milestone 3). The transformed tissues are being selected for herbicide tolerance.

Finally, in preparation for the second year of the project, we have also been investigating the potential of adopting a new homologous recombination protocol, named as CRISPEY, in the research of generating novel herbicide tolerance traits. We are also testing a novel base-editing Cas9 that would allow us to modify a longer stretch of genes.
Again, we wish to thank NCSRP for the renewal of our project, and for the confidence you placed with us!

Final Project Results

1. We have calibrated a streamlined protocol to quickly turn very young soybean seed into embryonic tissues in liquid cultures.
2. We have engineered the DNA constructs that once delivered into soybean cells, drive the so-called CRISPR base editing to modify specific soybean genes.
3. We used the two sets of tools outlined above to alter one of the soybean genes, known as GmALS1, at one of the amino acid positions.
4. We have succeeded in modifying the GmALS1 gene in a specific way that turned it into an enzyme that degrades the herbicide Imazapyr. This has been done in one soybean cultivar. The same set of DNA constructs are being tested in other soybean cultivars.
5. We are currently rearing the soybean seedlings in which GmALS1 has been modified in greenhouse, waiting for seed to set.

Benefit to Soybean Farmers

While we anticipate that we will inevitably encounter unexpected setbacks and obstacles, we are confident that our collaborative effort will allow us to achieve the goals and specific objectives set in the proposal. We are excited with the prospect that if our project progresses as anticipated, the results of our research will yield soybean varieties that are resistant to three novel classes of herbicides (targeting ALS, PDS, and HPPD genes respectively). These herbicides pose little safety issues because, although not widely used in soybean, they have been previously used in other crops including corn, and also to control aquatic weeds. These new soybean herbicide tolerance traits will allow farmers to control weeds more effectively, with greater consistency and cost-savings. This would in turn enable the growers to increase profitability from growing soybean.

Performance Metrics

For the first year of our project, spanning from October 1, 2018 to September 30, 2019, the performance metrics are:
(1) Successful base editing of the soybean ALS gene by using germinating soybean seed and particle bombardment DNA delivery, generating Imazapyr-resistant soybean seed;
(2) Successful assembly of DNA constructs for producing BE3 (base editor, third generation)-transgenic soybean;
(3) Initiation of soybean transformation to generate BE3-transgenic soybean in a number of soybean cultivars: Williams 82, Thorne, Maverick, Bert, and Jack.
We are proud to announce that all these metrics have been met. We are currently gearing toward another exciting year!

Project Years