This multistate collaborative 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-folds: (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, 2 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 use alone, in combination, or in alternation, helping them gaining the upper hand against the weeds in the soybean fields, and achieving higher soybean productivity in a sustainable manner.

(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.

(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.

Updated April 1, 2021:
Statement related to COVID-19 pandemic The COVID-19 pandemic and the related restriction to lab and office spaces has greatly slowed down the progress of our research. More specifically, it is preventing us from hiring highly qualified researchers to carry out the planned research. Students and visiting scholars from abroad are having enormous difficulties in getting the entry visa to join us.
We are trying our best to continue our research. However, as we have communicated with NCSRP earlier, we may not be able to complete a substantial portion of the proposed research due to staff shortage. We plan to return the unused funds back to NCSRP, and strive to continue our research in future years.

Specific progresses:
1. We have early success in developing 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. We are happy to report here that the initial results are telling us that we are successful.
2. We are adopting a new, more efficient base editor. 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. We have succeeded in putting the new base editor gene in a plant transformation vector. Next we will try to use this new base editor to generate transgenic soybeans.
3. We are attempting to engineer herbicide tolerance in soybean using a non-CRISPR approach. 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.

Updated October 26, 2021:
Progress Report:
Statement related to COVID-19 pandemic The COVID-19 pandemic and the related restriction to lab and office spaces has greatly slowed down the progress of our research. More specifically, it is preventing us from hiring highly qualified researchers to carry out the planned research. Students and visiting scholars from abroad are having enormous difficulties in getting the entry visa to join us.
We are trying our best to continue our research. However, as we have communicated with NCSRP earlier, we may not be able to complete a substantial portion of the proposed research due to staff shortage. We plan to return the unused funds back to NCSRP, and strive to continue our research in future years.

Specific progresses:
1. The novel, simplified soybean transformation protocol failed to lead consistently higher transformation efficiency. 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. However, while we have had some early successes, Upon more careful comparison this protocol failed to lead to higher transformation efficiency. While disappointed, we wish to note that such explorative research is inherently risky. Our experiences serve to warn other researcher against similar attempts.
2. We are adopting a new, more efficient base editor. 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. We have succeeded in putting the new base editor gene in a plant transformation vector. We are currently trying to use this new base editor to generate transgenic soybeans.
3. We are attempting to engineer herbicide tolerance in soybean using a non-CRISPR approach. 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. We have carried out extensive investigations on OsHIS1 in a transient expression system. Constructs for soybean transformation are being assembled, and will be used for transforming soybean very soon.

Updated April 6, 2022:
Statement related to COVID-19 pandemic The COVID-19 pandemic and the related restriction to lab and office spaces has greatly slowed down the progress of our research. More specifically, it is preventing us from hiring highly qualified researchers to carry out the planned research. Students and visiting scholars from abroad are having enormous difficulties in getting the entry visa to join us.
We are trying our best to continue our research. However, as we have communicated with NCSRP earlier, we may not be able to complete a substantial portion of the proposed research due to staff shortage. We plan to return the unused funds back to NCSRP, and strive to continue our research in future years.

Specific progresses:
1. The novel, simplified soybean transformation protocol failed to lead consistently higher transformation efficiency. This new protocol uses embryo axes isolated from germinating soybean seed, was thus expected to accelerate the production of transgenic soybean harboring the base-editing enzymes. This is also part of the goals of the project. However, while we have had some early successes, Upon more careful comparison this protocol failed to lead to higher transformation efficiency. While disappointed, we wish to note that such explorative research is inherently risky. Our experiences serve to warn other researcher against similar attempts.
2. We have adopted a new, more efficient base editor. 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. Upon resuming the base editing investigations after COVID-19 lockdown, we assembled new base editing constructs based on the more efficient BE3 design. These vectors are now being transformed to soybean plants in Dr. Clemente’s lab at University of Nebraska-Lincoln. Despite the termination of NCSRP funding, we remain dedicated to continued characterization of these transgenic lines.
3. We have engineered herbicide tolerance in soybean using a non-CRISPR approach. To provide soybean growers with more choices of herbicide tolerance traits, we have also adopted 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. We have carried out extensive investigations on OsHIS1 in a transient expression system. Constructs for soybean transformation have been assembled, and are currently been transformed into soybean in the lab of Dr. Clemente, at University of Nebraska-Lincoln. We will report back to NCSRP once the transgenic plants become available for testing.

Final Project Results (layman's terms for all audiences):
The COVID-19 pandemic-caused lockdown, and the severely restricted reopening that limits the number of people per lab to two at our working sites, have devastated our research in multiple ways. It completely destroyed all of the transgenic events we produced during the previous two years. More seriously, the COVID-19-caused freeze in hiring also prevented us from hiring the necessary staff until early 2021. All these challenges have prevented us from achieved many of the goals we set in the proposal.
Despite these challenges, we made substantial progresses in the following fronts:
1. We have learned that soybean transformation using embryo axes is not substantially superior than existing transformation techniques. This is a bitter pill for us to swallow as we have invested a substantial amount of our manpower and resources at developing this protocol. However, we also have to honest to acknowledge the failure when faced with the results. The lesson we learned is that we should be careful with trying the new approaches that some researcher claimed to work in research papers.
2. We have continued our effort of optimizing the procedure of using base editing to engineer novel herbicide tolerance traits in soybean. We adopted a new base editor, known as CBE4max-SpRY, for soybean use. While the long time needed for soybean transformation prevented us from assessing the efficiency of this new base editor within the funding period, we are determined to continue the research as soon as the transgenic plants are received.
3. We have begun to incorporate a new herbicide tolerance gene into soybean using a non-CRISPR approach. To provide soybean growers with more herbicide tolerance traits, we have adopted 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. The relevant constructs have been assembled, and sent to collaborators’ lab for generating transgenic soybean plants.

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.