When Dr. Tim Croughan was looking for rice that could be resistant to the herbicide imazethapyr, he tested thousands of mutant plants he developed in his laboratory at the LSU AgCenter’s Rice Research Station in Crowley, La.
After Croughan identified one resistant line in the early 1990s, it took a team of LSU researchers led by Dr. Steve Linscombe several more years to transfer the resistance into a Clearfield rice variety that could be grown commercially in the southern United States.
The odds are that process could be replicated in a fraction of the time with gene editing using CRISPR Cas9 technology, according to Dr. Michael J. Thomson, professor in the Department of Soil and Crop Sciences at Texas A&M University and holder of the Henry M. Beachell Chair in international rice improvement.
“If you look at what we’ve traditionally done with traits such as Roundup Ready or glyphosate-tolerant cotton and soybeans, our tolerance was provided by a transgenic approach,” said Thomson, speaking during a University of Arkansas System Division of Agriculture Food and Agribusiness Webinar (https://youtu.be/6IQTKu42Los).
“So now there are ways to do the modification of essentially the endogenous genes (which originate from within an organism, tissue or cell) so that you can use CRISPR Cas9 in what could be a non-transgenic approach to this.”
CRISPR or Clustered Regularly Interspaced Short Palindromic Repeats technology was first described in a paper in 1987. It was first proposed as a potential tool for genome editing in a landmark paper written by Dr. Jennifer Doudna of the University of California, Berkeley, and Dr. Emmanuelle Charpentier from France in 2012.
CRISPR and the CRISPR-associated protein 9 or Cas9, a protein found in the Streptococcus bacterial immune system, cooperate with guide RNA and work like scissors, attacking the DNA of invading viruses and slicing it up. By “knocking out” selected genes, it can also help scientists better understand the function of those genes or allow other genes to more fully express themselves.
That would be a far cry from the process used to develop Clearfield rice, which is non-transgenic. “In the past, using mutation breeding, it was still quite a long process to find the perfect mutation — essentially it’s a brute force method,” said Thomson. “You’re just making a lot of random mutations in the genome, and you’re hoping that you can get the right one for your desired effect.”
With the acetolactate synthase inhibiting or ALS class of herbicides, for example, research has uncovered some of the reasons why weeds become resistant to those chemistries in field settings.
“In this case, we can do a very targeted mutation,” he said. “With those amino acid changes we have a lot of data on what provides that herbicide tolerance, and we can use that make those modifications in our materials for breeding lines.
“Now, this is a little tricky in that you don’t want to overuse some of these herbicide tolerance genes because then, again, you’ll have the tolerant weed problem. But this could be used as a way to pyramid multiple mechanisms or modes of action of herbicide tolerance into the same line so you have, then, many options when you’re dealing with weeds.”
In another recent publication, scientists described using biolistics to modify the amino acids for ALS resistance in corn, using a method called ribonucleoprotein or RNP delivery. “What you see is that it does have the herbicide tolerance, provided by these specific amino acid changes.,” Thomson said. “But what’s interesting is that with RNP delivery, it’s essentially delivery of CRISPR Cas9. In the end, there’s no DNA that’s integrated into the genome; it’s essentially a DNA-free approach to gene editing. And it’s also essentially non‑transgenic, as well.
“This method uses a purified Cas9 protein with guide RNA mixture, and you make that complex in vitro before you shoot it into the plant cells. You’re starting with a Cas9 protein and the RNA complex. Then you introduce it using the gene gun, so biolistics can make that edit very quickly. It’s a transient approach.”
After a week or two, the protein will be naturally degraded by the cells, but that’s enough time for it to make the desired mutation in the target genome, Thomson says. “In this case, there’s no DNA integration of any foreign DNA, and this would be one of the more straightforward ways to obtain a non-GMO product.”
Thomson says there are new products being developed by companies such as Calyxt, using gene editing techniques. Among those are high-oleic content soybean oil, high-fiber wheat, powdery mildew-resistant wheat, anti-browning mushrooms, disease-resistant citrus, fungus-resistant bananas and reduced-gluten what. Growers may also have new crops to grow, crops developed from wild plants domesticated by using CRISPR technology.
Numerous legal and regulatory hurdles remain for the technology, although early indications are neither USDA nor the Food and Drug Administration are keen on regulating the products primarily derived through gene editing.
Based on the early work by Doudna, the University of California, Berkely, has become involved in patent litigation with such coalitions as the Massachusetts Institute of Technology and the Broad Institute.
“The medical field is the biggest area of application, but agriculture has gotten a lot of attention recently,” said Thomson. “A year ago DuPont Pioneer and the Broad Institute essentially joined forces to enable licensing of the technology for agriculture. It’s provided some clarity in that groups know who to talk to if they want to license the technology.”
For more information on the University of Arkansas webinar series, visit https://bit.ly/2qytJUu.