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Research Highlights

Research Highlights
Researchers Employing CRISPR for Soybean Advancement

By Carol Brown

In the biological research industry, there is a lot of buzz circulating around CRISPR/Cas, commonly referred to as just CRISPR. It is an emerging genome editing technology with great potential to make scientific breakthroughs across the medical and agricultural sectors. Sparking even more excitement, the 2020 Nobel Prize in Chemistry was recently awarded to CRISPR pioneers Emmanuelle Charpentier and Jennifer A. Doudna.

In 2012, Charpentier and Doudna published their initial paper on CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats. The pair were studying a bacterium that is harmful to humans and found that CRISPR could be applied to any organism’s DNA molecules. Researchers around the globe are now using CRISPR as a tool in genetic advancement for humans, animals and plants.

Chair of the Nobel Committee for Chemistry Claes Gustafsson stated in a Nobel Prize press release, “there is enormous power in this genetic tool, which affects us all. It has not only revolutionized basic science, but also resulted in innovative crops and will lead to ground-breaking new medical treatments.” 

CRISPR in infancy

In the greater scientific realm, CRISPR is quite vogue, especially in agricultural advancement. The first papers on its use with soybeans were published only five years ago in 2015 through projects funded by the United Soybean Board (USB). Researchers are using this tool with a number of checkoff-funded projects through USB, North Central Soybean Research Program (NCSRP) and various state soybean organizations. 

“CRISPR-based technologies are pretty new, but are advancing rapidly,” says Steve Whitham, a plant pathology professor at Iowa State University (ISU). “It seems like every few months there’s some new twist on the technology.”

What is CRISPR/Cas?

CRISPR is the acronym for Clustered Regularly Interspaced Short Palindromic Repeats, which works together with Cas (CRISPR-associated protein) as “molecular scissors” that cut one or both strands of DNA at certain locations to allow for improvements in plants and animals. It can help plants in many ways such as being more tolerant of disease or drought. In humans, it could help with genetic diseases such as Alzheimer’s, sickle cell disease or cystic fibrosis.

CRISPR/Cas technology is comprised of two parts: guide RNA and a Cas enzyme. Ohio State University Professor Feng Qu uses a metaphor of a road excavator to illustrate how it works. To fix a bad portion of a road, both an excavator and a GPS are needed, he says. With CRISPR, the excavator is the Cas enzyme, and the GPS is the guide RNA. Without the GPS, the excavator does not know where to dig. The guide RNA brings the Cas enzyme to the specific location in the genome. The Cas will only edit at this location.

Figure 1A. Cas9 nuclease is guided to a specific site on the target DNA by the specific sequences in the guide RNA (shown in green). After the target DNA has been cut, other cellular processes work to repair the damage. Small insertions or deletions (called indels) can be random or specific.

Figure 1B. If random, the mutation usually results in silencing or knocking out (turning off) protein expression from that gene. If specific, the indel mutations may regulate gene expression (gene regulation) for more or less protein. Other specific indel mutations modify genes to produce proteins that have new and better functionality. Finally, if novel genes are provided they may be inserted (gene insertion) into the cut DNA during repair for entirely new beneficial proteins. Graphic courtesy Iowa Soybean Association.

Scientists are examining how CRISPR can be used to achieve their anticipated results, and are defining how to make the overall process more effective for all scientists. Many researchers have included this component in their project goals.

“CRISPR has great potential, but there are still a lot of basic questions to be addressed,” says Ed Anderson, NCSRP Executive Director. “Some of the NCSRP funding is directed at just optimizing the CRISPR technology for soybeans because it’s not completely straightforward and easy.”

University of Minnesota agronomy professor Robert Stupar is collaborating on several projects in which CRISPR technology advancement itself is a goal.

“One project is with my colleague, assistant professor Feng Zhang,” Stupar says. “He has been developing methods to make gene editing platforms easier for soybean researchers. It’s more of an enabling technologies type of project, where we’re trying to develop ways to make the process more efficient and more useful for other people.” 

Understanding the soybean

At the University of Georgia, professor Wayne Parrott is also using CRISPR in his lab. His team is focused on adapting the technology with other crops such as switchgrass as well as helping to advance soybean biology. 

“Because the soybean genome is so large, conducting genetic research on the plant is challenging,” Parrott says. “Soybeans have approximately 50,000 genes and we don’t know what 40,000 of them do.” 

Researchers including Parrott, Whitham and Stupar continue to ensure that expanding our knowledge of soybean biology is a priority.

“One of our bigger challenges is that we need to have a strong understanding of how a gene is contributing to a given trait, which may be important to production, or quality, or other factor,” Whitham says. “As we’re developing the CRISPR technology, we also want to understand the genes and their functions better to make good choices about how to edit the gene and maximize the soybean’s potential.”

A multi-state group that Stupar is collaborating with use CRISPR in the role as a gene validation tool. 

“If we think a gene functions in a certain way, one of the best ways to validate that is to mutate that gene and see what happens to the plant. We use CRISPR for that,” Stupar says. “It’s a basic science question in which CRISPR enables us to discover what genes do.”

Applying the technology

Many researchers work with other plants first before transitioning to soybeans. At ISU, Whitham is conducting a project, funded by the Iowa Soybean Association, that includes using CRISPR to identify emerging genetic technology before applying it to soybeans. 

“We work with a model plant, Nicotiana benthamiana, a cousin of tobacco,” says Whitham. “The plant has a number of assets that are easier to work with than corn or soybeans. We like to try to prove a concept in the model plant, then move it into corn and soybeans.”

He is collaborating with University of Missouri plant sciences professor Bing Yang in similar study trying CRISPR applications first in another model plant, Arabidopsis (rockcress), a common plant that biologists use as a model organism.

Various ongoing checkoff-funded research projects use CRISPR to advance particular soybean traits such as improved seed composition and protein levels, preferred plant architecture, and increased pathogen resistance, to name a few. These projects entail finding unwanted traits and using the technology to “knock out” or remove the undesirable gene. 

“CRISPR can accomplish both jobs — getting rid of unwanted traits as well as adding in desirable traits, but deleting traits is better understood,” Parrott says, “Adding traits isn’t as straightforward.”

At Ohio State University, plant pathologist Feng Qu is leading a collaborative project, supported by NCSRP, with Stupar in Minnesota and researchers at the University of Missouri and the University of Nebraska. Qu and his colleagues are trying to add traits to soybeans to increase herbicide tolerance.

“CRISPR technology is very powerful because it can cause a precise change inside the genome,” Qu says. “We are trying to engineer the normal soybean trait for herbicide tolerance, and we want to use CRISPR to make changes so the plant can gain tolerance to other herbicides.”

Classical CRISPR technology is primarily used to make knock outs, Qu says, but that isn’t where his interests lie. His goal is to not just cut out an undesirable trait but to modify the previously functioning gene so that it maintains its original function and add tolerance to other herbicides, too. 

“We want to use CRISPR to improve gene function, not just knock out the gene. We’ve been working on this for three years and have had some success,” Qu says.

Miles yet to go

Although CRISPR-based technology will help speed up genetic research, it is not a silver bullet solution.

“Some people may perceive that it’s ready to go, it won’t require regulation, and products coming from CRISPR technology will be commercially available tomorrow,” says Anderson. “But that’s a naïve perspective.” 

Scientists need to think of CRISPR as another valuable tool, Stupar says, but it doesn’t replace plant breeding. 

“No one technology will solve all of our problems,” Stupar says, “but expanding our toolbox and giving us more ways to solve problems is as good thing.”

Parrott says that although it is still early in the technology, CRISPR beats the alternatives. 

“To describe CRISPR in a nutshell, it’s what we’ve been doing except it allows us to do it much more precisely and quickly,” Parrott says. “CRISPR has been wonderful to use as we try to determine what genes do, and in turn, helps us decide what to do in a breeding program, which is the ultimate goal. The generation that came before me had their entire career without any new technology. In my career, it seems like there is something new every five to 10 years. It’s an amazing ride.”

Selected Projects Involving CRISPR with Checkoff Funding

Gene Editing to Increase Soy Protein Content:
https://www.soybeanresearchdata.com/Project.aspx?id=53245

Genomic Tools to Enable Trait Discovery and Deployment:
https://www.soybeanresearchdata.com/Project.aspx?id=52897

Improving CRISPR Gene Editing in Soybean:
https://www.soybeanresearchdata.com/Project.aspx?id=52576

Manipulating a Major Gene Governing Seed Reserves as a Means to Maintain Yield and Oil While Increasing Protein:
https://www.soybeanresearchdata.com/Project.aspx?id=53685

Network Guided Modification of Soybean Meal Composition:
https://www.soybeanresearchdata.com/Project.aspx?id=52684

New Breeding Technologies Applied to Meal:
https://www.soybeanresearchdata.com/Project.aspx?id=52894

Non-Transgenic Generation of Herbicide Resistance in Soybean Using CRISPR Base Editing:
https://www.soybeanresearchdata.com/Project.aspx?id=53004

Platform for Fast and Cost-Efficient CRISPR Gene:
https://www.soybeanresearchdata.com/Project.aspx?id=53061