Gene editing, genetic modification, transgenic; ever wonder what those terms mean? Eduard Akhunov with Kansas State University does, and he’ll explain it all in this episode. Learn how powerful new tools like gene editing can transform the world of wheat breeding, and how science is leveraging ancient genetics to improve modern-day wheat.
00:00:02
Welcome to the Wheat's On Your Mind podcast. I'm Aaron Harrys. Wheat's On Your Mind is brought to you by the Kansas Wheat Commission and Kansas Association of Wheat Growers. Our guest on this episode is Eduard Akhenov with Kansas State University. Eduard Akhunov is a university distinguished professor of wheat genome diversity and pollution in the department of plant pathology here in Manhattan, Kansas. Eduard, thank you for joining us. I've been looking forward to this, Eduard, because we need to talk about the science of genetically modified organisms, and there's a lot of confusion that goes on around this. But you, of course, do a lot of research that is funded in part by Kansas wheat farmers through the wheat checkoff about gene editing. And we will get to that. But let's start just at the basics of what a person like you does. I know we've talked about this in the past that weep. Breeders will generally look at things like specific physical traits when they're crossing plants to create new varieties. But you're going even smaller. You're looking at genes. That's really what it's all about, right?
00:01:71
Yes, that's true. Maybe. One thing that I would like to tell about gene editing in comparison to more traditional existing technologies that are broadly used in breeding for wheat improvement is that gene editing is essentially just another way of generating new genetic diversity in wheat or in any other crop. And genetic diversity is one of the foundations of crop improvement. And breeders are always trying to bring new genetic diversity in their breeding programs, and they achieve it using various approaches. And for example, here at WRC, we also work with the wild relatives of wheat that we cross with wheat and then use this genetic diversity to improve wheat. CRISPR technology is just another way, but maybe a very precise way of modifying with genome and mostly focusing on modifying genes that are controlling specific traits. So that's probably the main difference, that it's precise and focus on individual genes.
00:02:145
Yeah, that genetic diversity is key in a huge tool, the WGRC, as you reference, the wheat genetics resource center here at K-State. And we'll talk about that in a bit. I think there's some confusion sometimes on what genetically modified. Know, let's, let's clarify that up for some people. So what most people call a GMO, a GMO corn or soybeans or GMO food, I think they're mostly referring to what's called transgenic. So what's the difference between transgenic and gene editing, which is like CRISPR cas nine is a form of gene editing.
00:03:181
The main difference would probably be that in case of transgenics. We are introducing into a crop or into wheat an alien gene that is not naturally present in wheat. And this gene should remain there for trait to be expressed so that we could actually modify what we are interested in. Whereas CRISPR editing is precise way of modifying specific part of the genome and leaving a small scar in the genome. But you don't have to have alien gene present in the genome for trade to be expressed.
00:04:222
In this case, for example, the GMO corn that is out there on the marketplace has a bacteria in it. That's that alien DNA or alien species you were talking about introduced into that makes it GMO with gene editing work. It's using the material that's already there without introducing something new into it. So your options are kind of, you can move gene parts around within that wheat plant, you can cut parts out and put them back together. Are those all different options that you have?
00:04:255
Yes. So, CRISPR editing provides very diverse set of tools for modifying genome. You could remove genes, you could put new pieces, or you could precisely modify a gene to improve the trait. But in all these cases, you are not introducing something new. You are essentially using the existing genetic code, but you are just tweaking a little bit to improve traits that you are interested in.
00:05:283
And the precursor tool to gene editing, if I understand it, was something called tilling, which is a form of creating mutations in the plants to kind of vary those genetics through mutations. But that wasn't nearly as precise, is that correct?
00:05:298
It is actually a random way of generating genetic diversity in the genome by treating seeds of crop plants with a chemical mutagen. And as a result of that, the new genetic diversity generated is spread across the entire genome. It's not very precise, but it is not regulated the same way as Gmo or genetic crops.
00:05:327
Do those kind of mutations happen in nature?
00:05:329
They do. They do. And process of mutations, actually constantly ongoing. Every generation, every plant would produce a number of mutations due to some imprecisions of copying DNA during the reproduction. And that's a natural process, and that's what entire breeding and crop improvement is actually relying on. Whereas with the gene editing tools, you could now introduce precisely mutations in the region of the gene that you think could improve the trait.
00:06:360
So we've kind of learned from nature, reverse engineered it in a laboratory, but just much more specifically. Yes, precisely.
00:06:367
Rather than looking for the natural diversity, we could also focus on specific gene once we know its function. Actually knowing function of the gene in this case is quite important.
00:06:379
Yeah, that is important. So we've talked about with other guests on this podcast, just on how complex the wheat genome is. A hexaploid plant. Six sets of seven different chromosomes, very complex. And so you have to know where these traits are before you can even think about editing them. But because it is a hexapoid, it takes more than one edit, right?
00:07:401
It does. And that's one issues with the regulation of gene edited crops at this point, which genome is gexapoid plant. It has three different genomes. And as a result of that, most of the genes present in at least three copies in the genome. And to achieve effect of editing that you need to edit all free copies.
00:07:424
Otherwise, the change doesn't take.
00:07:427
It will be less pronounced and expressed, and the best results usually you achieve while you are editing all copies.
00:07:435
Okay, well, speaking of the wheat genome, in 2017, the world announced a rough map, or maybe a first drap map, of the bread wheat genome. That was a project that took a decade or two to actually create that map. So where does that stand now, and how does it help you in your program?
00:08:457
As we discussed earlier, gene editing is focused on modifying individual genes and knowing the structure of the gene, where it is, how many copies of the gene you have. It's actually quite important to have precise genetic information about individual wheat varieties. If previously, sequencing of wheat genome took a lot of time and was huge international effort, now, modern technologies allow you to sequence genome at cost ranging from $10,000 to $20,000. And cost keep going down. Now, when you start new gene editing project, it is quite straightforward to take a variety and then generate all genome sequence data, assemble it, identify the structure of the genes that you would like to edit, and then, based on that design, precise gene editing strategy.
00:09:515
Okay, well, we'll talk a little bit here about the potential of what you can do with that tool. But let's jump into the technique itself, CRISPR cas nine. So, break that down to us. What is CRISPR and what is cas nine?
00:09:529
Well, CRISPR well, it has very long name clustered, regular interspaced repeats that won't.
00:09:539
Be on the test.
00:09:543
CRISPR cas nine technology relies on two parts, where cas nine is a nucleus that could cut DNA.
00:09:549
It's a scissors.
00:09:550
It's a scIssor. But for scissor to cut DNA in exact position, it requires so called guide RNAs. These are small pieces of rnas that could guide the CRISPR cas nine complex to a specific place in the genome, so that the modification would not impact any other part of the genome, but will be done in a precise location.
00:10:577
What are you doing with that scissors? And you direct it with the rna. So if you make a cut to knock something out or put something in. How to.
00:10:587
Well, once the cut is created, you could do a lot of different things with this cut. And the easiest way is to knock out the gene so that it could lose the function and in some cases, actually beneficial, because there are some genes that negative regulators and negatively impact traits. And, for example, some of the genes that we have edited in the past are genes that are controlling grain size, and we know some negative regulators of grain size and grain number that we could knock out. And then their loss of function in these genes would improve the trait. But on top of that, you could also modify genetic code in the region of the cut, or you could introduce a small piece of DNA there that will have the genetic code that will be beneficial for that. Right.
00:11:638
It boggles my mind, and I'm sure most people's mind is. The question they probably have is, well, how do you insert that little piece in, move it from another region? It's all about that RNA, that guy.
00:11:650
Well, it's about rna, but then also you could provide a short piece of DNA along with, in your experiment, you could provide, along with the CRISPR cas nine, construct itself a piece of dna that you'd like to place into this. Just introduce this region of the genome.
00:11:669
I see. So the traits, I think one of the fascinating things you've taught me is that some of these genetic traits are the same across species, is that correct?
00:11:679
Yes, that's probably one of the advantages now we have with the CRISPR technology. CRISPR technology is the one that could actually utilize all the knowledge that crop geneticists accumulated over decades of their research. And in some cases, researchers would investigate yield traits in rice or sorghum or maize and identify genes that control this trait. But then comparison of the genomic data now available for all these crops allows to compare across the species and identify identical genes among the crops. And in this case, I could use the information collected by my colleagues from other crop communities to transfer that into wheat and then try to modify the gene using CRISPR technology. And in many cases, it turned out that the genes that we modify have similar effect or affect the same trait that was characterized and studied in other crops.
00:12:740
Well, speaking of other crops, you mentioned WGRC, which is the wheat Genetics Resource center, which is housed, actually here in the wheat Innovation center in Manhattan. But that's been at K-State for, I think, 40, maybe more years now. It's a collection of seeds essentially from wild relatives of bread wheat, the ancient grasses that created breadweat over several millennia, and that collection bringing in that diversity into modern wheat lines. How does that happen?
00:13:773
Oh, that's a little bit outside of CRISPR talk, but this is a multi stage process and requires diverse expertise. Usually, transfer starts with making crosses between wild relative and wheat, and then developing special genetic stalks, lines that would carry short pieces of the wild relative chromosomes that are fused with the chromosomes of wheat. And once this is done, then we try to screen newly developed lines for the traits that would like to improve in wheat, which might include disease resistance, quality, yield, adaptation to drought stress or heat stress, and then work with special genetic lines that carry mutated versions of the gene that control recombination between wheat chromosomes and the wild relatives chromosomes. The problem usually lies in the biology of wheat, because wheat chromosomes don't like to recombine with wild relative chromosomes because of the gene called ph, one that controls recombination between wheat chromosomes and wild chromosomes. And we have special genetic stocks where this gene is mutated. And this specific process, we could actually now could promote recombination between wheat and wild relative chromosomes and identify so called recombinants that will carry shorter pieces of wild relative chromosomes that then could be used by breeders in their breeding programs.
00:15:876
So, does gene editing or CRISPR cas nine in that situation, does it have application on the wild relative first or after the recombination has been made, or both?
00:15:887
There are a lot of intriguing opportunities to use CRISPR technology in combination with wild relative based breeding. One of them could be to try to induce recombination by breaking wild relative chromosomes, creating breakpoints in wheat and then wild relative chromosomes, and hope that in this case, they could more easily recombine with each other in some crops, it was actually demonstrated that it works, but it hasn't been tested in wheat yet. So another option is to actually use CRISPR technology to modify genes in wheat that are controlling recombination between wheat and wild relative chromosomes. And there are many genes that are involved in that besides known ph one gene. There are several genes that have been identified that actually controlling this process, recombination between wheat and wild relative chromosomes. By applying CRISPR technology, we may introduce, we may modify these genes and then in the future help to promote recombination between wheat and wild relative chromosomes. Importantly, that's actually quite important step because it would accelerate efficiency of introducing wild relative diversity into breadwick. And then instead of spending several years developing this new genetic material with the new genetic stocks, we might accomplish it within a year.
00:16:981
And that is important because traditionally, combining those wild relative traits, which is a source of a lot of our disease resistance, it takes a long time. But if this could make it more efficient, that would certainly be helpful.
00:17:993
Yes.
00:17:993
So let's give an example that you've shared with me before about a specific trait that you've done with casper. And it's this taGw two, which is a size regulator in rice. So then how did you identify the same thing in wheat?
00:17:1012
There is a gene called GW two. It was identified in rice first time. And then it turned out that it controls grain size associated with increased yield in rice. And analysis of biology of this gene showed that it is negative regulator of grain size. In cases when you have reduction in the function of this gene, or you knock out this gene so that it loses its function, grain size increases substantially, up to ten to 20%. And we have identified similar gene in wheat and then conducted editing using CRISPR cas nine technology. Not to our surprise, of course, we hope that has similar function in wheat. This gene editing resulted in substantial increase in grain size. It was not associated with the negative impact on yield. On top of that, it turned out that the editing of gw two gene in wheat also improves protein content in grain. So there is another benefit of this type of editing. So this is actually this specific case shows that how you could remove from wheat genes that are negatively impacting the trait that you'd like to improve, which could be easily achieved using CRISPR technology.
00:18:1095
Yeah, I'm looking at a picture that the listener doesn't have the benefit of looking at, but lined up the same number of wheat kernels end to end, side to side. And by removing that regulator, it does make the kernel quite a bit bigger. And you can see that evidence here in the bottom.
00:19:1110
Yes, you have bigger grain. It's more plum.
00:19:1112
That's actually beautiful grain, which would be great for flower millers. They'd love to hear that. But of course, as you mentioned, the holy grail of hard red winter wheat is if you're going to increase the grain kernel size and the test weight, you want to increase the protein at the same time.
00:19:1127
Yes, you do achieve that. It turn out that the protein content does increase with the editing of this gene.
00:19:1134
Well, since we're on the topic of protein, which I think most people know in wheat is gluten, or gluten is formed with the proteins in wheat. Gliadins and glutenins combine with water and make gluten, which is what gives wheat that magic quality that we all love, making bread rise and everything like that. And Eduard, you've just had a very exciting paper published talking about editing wheat to reduce the immunoreactivity of wheat gluten on people with celiac's disease. And just as a refresher, people with celiac's disease can't digest properly the gluten that's in that bread wheat. So this is quite a breakthrough. Tell us how it came about and what it does.
00:20:1178
Well, this project actually started with the idea that with all these new genomic resources that we have now available, can we identify within the gluten encoding genes? And we specifically focused on the class of genes called glidins. Can you identify those copies of glidian genes that carry the largest number of immunotoxic peptides? These short immunotoxic peptides are the cause of celiac disease. So in many cases also they are associated with other gluten sensitivity issues. Recently released whole genome sequence of one of the wheat varieties allowed us to perform very comprehensive analysis of immunotoxic peptides across all copies of gliding genes. And based on this analysis, we have identified those that have the largest number. Then obviously this should be contributing most to this immunotoxic reaction that you get from gluten.
00:21:1245
So how many glidins are there? I mean, is it dozens, hundreds?
00:21:1249
Well, it's in the order of several dozen. It varies from variety to variety. And there is a lot of variation in the dna sequence, in the sequence of these genes. So that's why it was actually quite important to have access to the genome sequence of the variety that you would like to edit.
00:21:1269
Right.
00:21:1269
And then that was probably one of the advantages that we had compared to the research that was previously done in that area. And then we designed CRISPR cas nine guides that would be targeting only these copies of genes. And we focused on only two classes of gliding genes, gamma and omega. They actually represent smaller fraction of all the glidians, because the main class of glidians are called alphabetaglidians. They have been edited and characterized before. But we decided to take a look what we'll achieve if we'll focus on other classes of gliding genes, which actually also carried the largest number of toxic peptides. And to our surprise, we actually discovered that by editing only these copies, we could achieve much more dramatic decrease in immune response based on the analysis with specific antibodies. But other positive aspect of that research was that, surprisingly, we found also that protein quality that is important for dough making and bread making didn't really change much. It was even actually improved. So essentially, we kind of achieved these two objectives that actually somewhat contradictory in previous research, it was actually shown that attempts to reduce immunotoxicity of wheat using biotechnological approaches quite often lead to decreased protein quality. And then bread making properties of dough developed from this flour would actually lower. But in our case, we actually didn't see that we even seen some improvement in quality of dough.
00:23:1374
That prompts another couple of questions for me. First, on those glidins that you did edit, what exactly was the edit? I mean, in a sense, to try to silence them?
00:23:1385
Well, we essentially were trying to delete remove of this particular copies of glidins from the genome. Okay. And we achieved that. And then to actually confirm that we have done it, we took the gene edited line and then we sequenced its entire genome again.
00:23:1404
Okay.
00:23:1404
And then we compared it to the genome of the released variety. And then that's how we know what we removed what is still there. And then we're actually happy to see that targets the guide rnas that we designed for editing these glidian copies. They actually achieved exactly what we wanted to. We removed nearly all omega Gladians, which we removed half of the gamma glidians. With that, we also removed a lot of toxic epitopes that were within these genes. And then when we start running these assays to test the immune reactivity of protein from the flower, we have seen such a dramatic decrease in immunoreactivity that essentially we hope that we'll achieve that. But we have been quite impressed that it did happen.
00:24:1453
So you sound a little surprised by some of that. I mean, there's still a component of.
00:24:1456
Guessing and hoping, because gliding genes are quite complex gene families. You have so many copies of them, and then achieving exactly what you hope to do with such a complex and large genome. And the complex gene family is actually.
00:25:1474
So the second question to elaborate on this is, well, we're familiar with testing the baking qualities of wheat by having some actual grain milling into flour, even if it's a small sample, and baking it. And you're telling us, well, you know, that the protein quality increased.
00:25:1491
How do you know? Well, there are certain biochemical assets that you could apply. So we, for example, looked at gluten macro polymers that are formed when you mix flour with water. And then we run this biochemical assays to show that the gluten macropolymeric complex is actually large. And then it's actually improved compared to the line that we didn't edit. So that was one thing. But on the other hand, we actually run mixograph and ferinagraph assays on the dough that we made out of the flour of this line. So, essentially, we did run actual dough test, not only biochemical assay. We started with the biochemical assay. We knew that it should be better, but then breeders actually told us that, well, you actually need to go and then try to run more standard classical assays. And we did the dough mixing assay. And actually, everything worked out fine. It doesn't.
00:26:1553
So you edited enough wheat and actually grew some in a growth room, or.
00:26:1557
We grew in greenhouse, because we can't. Well, first of all, it's a spring wheat, but the second, we can't grow. Well, we need to still reach that stage when we could actually grow it in the field.
00:26:1568
That's the easy part.
00:26:1570
Well, that's the easy part. And then another thing, the actual testing of this project will probably happen in the future, because what we showed in our project, that we could actually generate new variants of gliding genes that have lower toxicity, but also improve dough quality. So we need to transfer these genes that we edited into harder at winter wheat, and then grow this wheat in the field, and then perform actual bake testing and to see what will be the impact on the final wheat products.
00:27:1611
So, how long does something like that take?
00:27:1613
Well, with the modern approaches and technologies, probably it may still take a couple of years before these genes will be transferred and grown in sufficient quantity in the field across multiple locations.
00:27:1630
It takes time.
00:27:1631
It will take time.
00:27:1632
So, in regards to the level of reduced toxicity, I don't like to use that word, but for people with celiac's disease, it is toxicity. So what percentage of reduction of that for people who have celiac's disease did you find here?
00:27:1647
Toxicity of gluten is actually quite complex trait by itself. It's defined by many components of gluten. If we look, because glutinins, they're also causing some reaction if we focus only on the gliding component. We achieved 47 fault reduction in immunotaxicity of the glidins. What it means for people with celiac disease in its severe form, of course, would certainly not recommend using this flower just and say that it is safe for celiac patients. But it is important step forward. What we know about glidins is that, yes, we could achieve. We could edit them, we could substantially, dramatically decrease their immunotoxicity. For people who didn't develop celiac symptoms yet, they may have mouth sensitivities, maybe some of the products developed using this wheat will be actually quite safe, but that will require more research in the future. And also, it actually showed us that, well, sometime in the future, we have potential to develop wheat lines that will be safer for people with negative response to gluten. And it may, probably will not happen very soon, but within maybe matter of ten to 20 years, we might actually see something happening in this area.
00:29:1736
It may be in conjunction with work that the pharmaceutical industry is doing. Combine those two things, it may change life for people. I know there are farmers who listen to this podcast who have family members who have celiac disease. They grow wheat, but they can't eat it. And that's very sad. And I think most people would like to eat weed if they could. So that's an amazing breakthrough. And the power of the gene editing tool is kind of demonstrated there. So what is the other potential? I mean, we've talked about the edits that you made here and making grain size bigger and increasing again the quality or quantity of that protein. What's within the reach of gene editing as a tool?
00:30:1780
That's probably a question that we'll be answering for next decade or so, what we could achieve and what we cannot. But I think that we should consider CRISPR cas nine technology as a very powerful tool in the hands of scientists. First of all, it helps us, actually, to better understand how genes function, and then which genes are involved in controlling agronomic traits that we care about. That's probably first thing. And second thing, probably we could easily, there are certain things that we could easily achieve with gene editing technologies. For example, we probably will see very soon wheat lines that will be resistant to various diseases. Applying gene editing technology for improving disease resistance is probably quite straightforward. Disease resistance, in some cases, it's a quite simple trade. For example, there are genes that are called so called susceptibility genes that make wheat more susceptible to disease. And by applying gene editing, it's quite easy to remove these genes from the wheat genome, which will make plants resistant. And this is happening now, and there are a number of projects that are moving in that direction. So for that specific trait, we'll probably see results very soon. With the reduced immunotoxicity and reduced allergenicity of wheat, I think that we'll also see some improvements. Probably everything would depend on how complex the trait is. And then for simple traits, we'll see simple trait. What I mean by that is that the trait is controlled by few genes with the strong, clearly defined effect on that trait. In this case, we could easily achieve the outcome by applying gene editing for more complex traits that, for example, drought tolerance, heat tolerance, maybe some of the yield component traits, which are controlled by a large number of genes. Progress will be probably slower, but it will be slow in the beginning, as with geneticists and crop geneticists, they actually work hard on figuring out what is the genetics of this trait, which genes are involved, and then, in parallel, gene editing technology itself is developing. And then I believe in the future, we could apply quite complex gene editing strategies and then modify multiple genes at the same time and achieve something that we cannot imagine. Now. Certainly, new gene genome sequencing technologies that allow you to easily sequence genomes, new data analysis technologies based on artificial intelligence, will help us to identify those genes that are controlling traits and choose the most optimal gene editing strategy. So all these things will come together in the future, and then we'll see something that maybe right now, we cannot imagine.
01:33:1969
The next ten years are going to be really exciting, aren't they? And when we research, that's good for sure. We're glad to have a guy like you around. We have to talk a little bit about regulation. I mean, gene editing is regulated by multiple agencies, FDA, EPA, USDA, just much as genetically modified or transgenic would be. And we've run into some challenges with that. What are those specific challenges?
01:33:1995
In some countries, genetic products, actually, there is a positive outlook in the sense of regulatory landscape here in the United States, I think quite early, we have actually quite also positive outlook. But one of the issues now we're dealing with is that wheat is polyploid, and then most of the genes are present in multiple copies. In Breadwick, we have free copies of almost of most of the genes. And to achieve the best outcome in gene editing experiments, we need to edit all three copies. Whereas the current regulations are actually allowing to conduct field trials without lot of approval for gene edits only in a single gene certainly benefits crops, more simple crops, crops with the simpler genomes, like rice, for example. But certainly it has negative impact on wheat research, but it just increased the time that is needed to go from the lab to the field and then collect the data that we need, making.
01:35:2072
Only one edit at a time. I think people would be curious to know some of the other hexaploid plants. I think strawberries.
01:35:2080
Well, strawberries are octoploids.
01:35:2082
Octo.
01:35:2082
Okay, so the best ones, the melons that we see in the stores.
01:35:2087
Yeah. Sugar beets, I think.
01:35:2090
Yeah, sugar beet. Oat. There are a number of crops that are actually polyploids. And then I think that regulations need to be adjusted to take this into account because we now do know a lot about the biology of polyploid crops. We do know that many genes that are redundant and duplicated in these polyploid crops, they actually have similar function. And to achieve the best outcome, we need to edit more than one copy of the gene.
01:35:2117
And I know you've been working with us and us wheat associates to try to work with regulators on that. So hopefully we get an optimistic solution to that. Well, Eduard, this has been a great discussion. It's a very exciting time with this technique and these tools, and you've made some great breakthroughs, and I'm sure we'll visit many times here in the future.
01:36:2138
Well, let's hope so. And looking forward to talking with you again.
01:36:2142
All right, thank you, Eduard.
01:36:2143
Yeah, thank you.
01:36:2151
Thank you to Eduard Akhenoff with Kansas State University for joining us on the Wheat's On Your Mind podcast. If you have questions or comments about this episode, email us at podcasts@kswheat.com I'm Aaron Harries. Thanks for listening.