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.