Project Details:

Title:
Non-transgenic generation of herbicide resistance in soybean using CRISPR base editing

Parent Project: Non-Transgenic Generation of Herbicide Resistance in Soybean Using CRISPR Base Editing
Checkoff Organization:North Central Soybean Research Program
Categories:Weed control, Breeding & genetics
Organization Project Code:NCSRP
Project Year:2020
Lead Principal Investigator:Feng Qu (Ohio Agricultural Research and Development Center)
Co-Principal Investigators:
Keywords:

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

Our project strives to address the challenges posted by the weeds in North Central soybean fields, which are becoming increasing hard to control, primarily due to the spreading of weeds resistant to herbicides currently usable on soybean.
The rationales for this project are two-fold:
(i) By equipping soybean with new tolerance traits against herbicides not currently used on soybean, use of more herbicides with diverse action modes on soybean will be made possible. Furthermore, two or more herbicides could also then be combined to delay the emergence of resistant weeds.
(ii) CRISPR-based gene editing, especially base editing, allows the creation of new herbicide tolerance traits by introducing precise changes into a select set of soybean genes. Unlike older herbicide tolerance traits, such as Round-up tolerance and Dicamba tolerance, this new technology does not introduce non-plant genes, thus could lessen the regulative burden associated with GMO.

Our specific objective for this project is to use the new CRISPR editing technology to modify three soybean genes – ALS, PDS, and HPPD. This would enable soybean to tolerate the corresponding herbicides that target them – e.g. Imazapyr, fluridone, and mesotrione. All three would be new traits in soybean and these three classes of herbicide are considered relatively safe. Upon successful completion of this project, we expect to deliver soybean farmers with more choices of herbicides that can be used alone, in combination, or in alternation, helping them gain the upper hand against weeds in soybean fields and achieving higher soybean productivity in a sustainable manner.

Project Objectives

(i) accelerate base editing in soybean by using germinating soybean seed;
(ii) streamline the base editing protocol in multiple soybean cultivars by equipping them with the base editing enzyme BE3;
(iii) generate novel herbicide resistance traits in soybean using the new base editing approach.

Project Deliverables

(i) A fast, cost-effective, and non-transgenic base editing protocol for accurately modifying soybean genes without disrupting their functions;
(ii) Multiple soybean lines resistant to diverse herbicides generated with the new base editing technology;
(iii) 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.
Together these deliverables will directly benefit soybean growers by increasing the profit of growing soybean while reducing inputs.

Progress of Work

Updated July 28, 2020:
By now we have completed the first 18 months of this NCSRP-funded project. We are proud to announce that we are well on our way to meet our goals set for the second year. However, as I have communicated with the NCSRP leadership a few days earlier, the current COVID-19 lockdown is causing severe interruptions, and potentially irreparable damages to our progresses. Below are the specific progresses we made so far.
• During the first year, 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.
• That construct, along with a control construct, has been used to transform embryogenic tissues generated from young soybean seed, using a particle bombardment procedure. We obtained five (5) transgenic events for the base-editing construct, and one (1) event for the control construct.
• The transgenic tissues were further induced to produce transgenic soybean seedlings. Three transgenic plants were successfully transplanted to soil for seed-setting. Additional transgenic tissues are at various stages of root and shoot inductions.
• During the first six months of the second year, we made further progress on two new fronts: (i) A new construct that encodes the rice HIS1 gene has been assembled and transformed into soybean embryogenic tissues. The HIS1 is an alternative strategy for the previously proposed strategy of base-editing the soybean HPPD gene, primarily because the soybean HPPD gene turns out to be difficult to modify with base-editing. (ii) A base-editing construct for soybean PDS gene has also been designed, and parts of this construct await synthesis by a commercial vendor.
• The soybean tissues transformed with the rice HIS1 gene are being propagated for seedling induction.
• We hasten to note that due to the COVID-19 lockdown, we are in danger of losing the materials we produced so far. We are trying our best to rescue these materials, and will report to NCSRP as soon as we are able to assess the losses.

Updated September 24, 2020:
Statement related to COVID-19 pandemic:
We were making steady progresses with all three of our original objectives before the COVID-19 caused our campuses to lock down. The lockdown was particularly devastating at the Ohio State University, the primary performance site of our research. We were prohibited from entering research labs from March 19 to early June, for more than 10 weeks. Although we requested for special exemptions to reenter the lab as soon as such exemptions became available in June, by then it was too late for us to rescue most of the transgenic soybean tissues we generated over the last year. Inability to replenish the culture media with nutrients and selection agents caused most transgenic tissue cultures to die out. Other tissues became non-regenerative calli that lost the ability to develop into seedlings.
We are currently trying our hardest to restart the production of transgenic materials, even though we are allowed in the lab only 50% of time. To adapt to the challenging research environment, we are now concentrating much of our efforts on developing a simpler soybean transformation protocol for producing soybean plants carrying the base-editing Cas9 protein. As you are probably aware, we previously took advantage of the soybean transformation protocol available in Dr. Finer’s lab, involving embryogenic tissue culture and particle bombardment delivery of transgene DNA. However, the COVID-19-caused loss make us acutely aware of the shortcomings of that protocol. Namely, the frequent media exchange and close monitoring of the cultured tissues demanded by that protocol made it extremely challenging to maintain healthy transgenic materials, given the limited access to the specialized lab space. By contrast, the new protocol under development uses germinating soybean seed as the starting material, and requires only a short period of tissue culture (six weeks). If successful, this new protocol will be much simpler, technically less demanding, and also substantially shorten the time needed for producing transgenic materials.

Specific progresses during the last six moths are:
1. We have initiated the development of a novel, simplified soybean transformation protocol. This new protocol uses embryo axes isolated from germinating soybean seed, is thus expected to accelerate the production of transgenic soybean harboring the base-editing enzymes. This is also part of the goals of the project.
2. We have incorporated a new, more efficient base editor in a plant transformation vector. This new base editor, known as CBE4max-SpRY, is the latest version of base editing Cas9. It has been shown in animal cells to be much more efficient than BE3, the base editor we initially used. Now that we have to restart most of the base-edited soybean lines anyway, we decide it is best to adopt the most advanced base editor. Right now we have succeeded in putting the new base editor gene in a vector. Upon sequence verification, the new construct will be used to transform soybean.
3. We are in the process of engineering herbicide tolerance in soybean using alternative approaches. To provide soybean growers with more herbicide tolerance traits, we are also trying to adopt a rice herbicide tolerance gene in soybean. This rice gene, known as OsHIS1, was recently found to confer tolerance to a class of herbicides known as HPPD inhibitors, such as mesotrione or MST. Although this would involve generating transgenic soybean, the source of the OsHIS1 gene is another food crop (rice). If successful, such transgenic soybean will serve as an alternative to the base editing approach, especially given the uncertainty related to COVID-19.

Performance Metrics:
For the last six months, spanning from April 1 to September 30, 2020, the performance metrics are:
(i) Successful base editing of the soybean gene PDS, generating soybeans resistant to the PDS inhibitor Norflurazon;
(ii) Growth and harvesting of BE3-transgenic soybean seed for all cultivars transformed (Williams 82, Thorne, Maverick, Bert, and Jack);
(iii) Characterization of the soybean HPPD gene in preparation for developing soybeans resistant to the HPPD inhibitor Mesotrione.
As we explained earlier, we regretfully report that we will have difficulties to meet these performance metrics, due to COVID-19-related losses and delays. We have hence requested a six-month, no-cost extension of the project. We are fully committed to meet these metrics at the end of the extension.



Final Project Results

Benefit to Soybean Farmers

The outcomes of this project, should it proceed as planned, will be soybean seed stocks equipped with three new herbicide tolerance traits, which can be separate or combined in the same seed. The immediate benefit to soybean growers is to allow them to use three novel classes of herbicides that are so far only used on other crops. Combination of these three traits, and also in combination with existing herbicide tolerance traits if desired, is expected to greatly broaden the herbicide choices for soybean growers, providing them with far greater flexibility and high effectiveness in weed control. Improved weed control is in turn expected to lead to higher soybean yield and productivity.

Performance Metrics

(i) Successful base editing of the soybean gene PDS, generating soybeans resistant to the PDS inhibitor Norflurazon;
(ii) Growth and harvesting of BE3-transgenic soybean seed for all cultivars transformed (Williams 82, Thorne, Maverick, Bert, and Jack);
(iii) Characterization of the soybean HPPD gene in preparation for developing soybeans resistant to the HPPD inhibitor Acuron.

Project Years