Updated April 1, 2021:
Statement related to COVID-19 pandemic The COVID-19 pandemic caused an initial lockdown of more than three months at our universities, followed by severely restricted reopening that limits the number of people per lab to two. The restriction is still ongoing as of this writing.
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 mid June of 2020. It completely destroyed all of the transgenic events we produced during the previous two years, and were still in tissue culture stage, as the lockdown prohibited us from entering the tissue culture room to carry out exchanges of culture media.
More seriously, the COVID-19-caused freeze in hiring also prevented us from hiring the necessary staff until early 2021. As we described in early reports, all these challenges have caused serious disruption to our project.
Due to the continued restriction in use of certain lab spaces (e.g. Dr. John Finer’s tissue culture lab) and equipment, we have been focusing our recent efforts primarily on developing an alternative soybean transformation protocol that can be carried out without substantial tissue culture, simpler and cost-effective, and most importantly takes much shorter time to get transgenic seeds. The preliminary results suggest that our efforts are starting to lead to concrete results.

Specific progresses are:
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.


Performance Metrics:
For the last funding period, which lasted 18 months including a COVID-19-related no-cost extension of six months, 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 have yet to fully meet these performance metrics, due to COVID-19-related losses and delays. However, we are adjusting our goals and making progresses to develop a new soybean transformation protocol that is less technically demanding and takes shorter time to get transgenic plants. We anticipate that the new protocol will allow us to final attack the base editing goals next.

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 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.