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.

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.

(i) Successful base editing of the soybean gene PDS, generating soybeans resistant to the PDS inhibitor norflurazon;
(ii) Development of a novel, simplified soybean transformation procedure that allows for more widespread adoption of CRISPR technology in soybean;
(iii) Development of transgenic soybean that express the rice gene HIS1 conferring tolerance to the HPPD inhibitor herbicide mesotrione (MST).