361 Precision Insect Control with Gene Editing
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Kevin Folta: [00:00:00] Hi everybody. And welcome to today's talking biotech podcast by collabrate. Now there's no question that the most dangerous and damaging animals on the planet are insects. Top of the list from spreading infectious disease to overnight devastation of crops, insects cause massive losses in human health.

They threaten food security and contribute in a big way to agricultural losses on the farm as well as food. So for more than half a century, scientists have used sterile insect technique to limit deleterious populations. It it's a pretty easy concept you capture or, or raise a bunch of insects like mosquitoes, and then you treat them with radiation.

You break chromosomes, you STR scramble, genetics, whatever. Lots of 'em die, but the ones that survive can be released and then introduced into a resident population, which are typically invasive insects. So you, you, you introduce the [00:01:00] defective dead end genetics to mate with the ones you wish to suppress.

The next generation is not as viable and populations usually. now all of this is good, but today, instead of using radiation and random damage of DNA, we can use much more surgical techniques. And in the past, I believe maybe couple years we've talked about this different transgenic techniques have been implemented.

We've talked about them on the podcast, mosquitoes fall armyworm Diamondback, moth, really, really great in. But a recent paper took an innovative approach using gene editing to suppress the levels of an agricultural pest. We'll talk to a representative from that project today. On today's podcast. Our guest is Dr.

Nicki condo. He's a project scientist in the cell and developmental biology department at the university of California, San Diego. So welcome to the podcast, Dr. Condo. Hello everybody. Yeah. So [00:02:00] the work that you did and your team did really, we revolved around. A creature called DSO Suzuki eye and more commonly known as spotted wing DSO.

Drosophila means a lot to some people, but not to others. so what is this thing in common terms and why is it a problem? 

Nikolay Kandul: Yeah, Zo Suzuki. I it relatively closely related to Zoga, which is essentially lab workforce for many scientist for has been lab workforce for more than 120 years. But. It's DSO species.

It's native from south east Asian Asia region. But it's a, it's a crop past. So it makes close pH connections to DSO. Non gastro makes it a very good object because we can test system in DSO and then we can port them into DSO, Suzuki and see if actually work in the past species.[00:03:00] 

Like we did with our. . 

Kevin Folta: Yeah. And so the, the big question I was kind of getting there is how would people recognize this? And this is really just a, a relative of the fruit fly. This is a kind of fruit fly, correct? Correct. Yeah. And so you're in Southern California. So why is this a big problem for farmers and for different industries?

Nikolay Kandul: This species is, as I said, native for Southeast Asia, but in 2018, it was sported in Hawaii and essentially sampled in Hawaii. And in 2008 it was Essentially found here in California. And then in 2010, it spread to Oregon and Washington state. And in 2012, it's essentially almost everywhere in us, including, and the life cycle was relatively short.

And in Japan during the summertime you can have 13 generations of Zo Suzu. So that makes [00:04:00] it very prolific and very bad pass because it's propagate so fast and it essentially different from DSO because females orzo Suzuki, they have rated with. With that of repositor they can insert the eggs into what is called soft skin, summer berries, like cherries, blueberries, raspberries, Blackberry peaches, nectarines, a lot of and because they can propagate so fast giving short lifestyle, they're very hard, has to deal.

Kevin Folta: Yeah, we have them here in Florida and there are substantial issues with, especially in strawberries. Seeing more and more in blueberries where spotted wing GESO is a formidable pest and farmers are constantly monitoring. And sometimes treating when they see it, it's really become a, a, a, a battle.

And so how has this been traditionally managed on the. 

Nikolay Kandul: It's hard to say because I mean, it it's relatively recent problem in California since [00:05:00] 2008. But right now farmers use traps to trap them and monitor. And if they see substantial infestation, they essentially have to use pesticide. For insecticides and they try to use them kind of in the morning, then pollinators are still not available.

That's when the flies are actively searching, like female flies actually searching for fruits to lay eggs, but it's you have to spread pesticides. Then you have fruits already ripening, and that's a big problem. 

Kevin Folta: Yeah. So you see that many different angles here. You're spraying fruit when it's ripe, which yeah.

You know, consumers are not thrilled about. You're also putting pesticides into the environment during a time when pollinators can be active as something else we don't like to do. So what we really need is a better solution to do this. And Maybe sterile insect technique. So we've talked about sterile insect technique on the podcast, as it relates to things like fall army worm to mosquitoes, to diamond back moth.

How [00:06:00] has sterile insect technique been used traditionally. And can you explain that process? 

Nikolay Kandul: Yeah sort of sterile insect technique was used in us since 1950s and probably the most canonical species where steroid insect technique was used most effectively. It's a new world. Screw won't. That fly essentially was devastating.

A cattle industry. That's a fly which essentially lay eggs in the skin of cows and then a will hatch and lobby will borrow into the live tissue of the cows and will eat that live tissue until it develops and leave. And so it was obviously big problem and they were common for seven states in us and downwards all central America and south America.

And I think in 1930s, it was the first time in Syria, broski back in Russia, start thinking that if you release sufficient number of [00:07:00] sterile insects into population, those insects were made And then you have a lot of effects, which don't hatch and you'll have some way to suppress population. And then that essentially theoretical research was picked up in us.

And it was applied for new world school flies and essentially how it's done UMass rare these flies in factories, then you Irradiate rose flies in a scuba fly at the pupil stage. You use radiation essentially to sterilize males, and then ideally you also want to remove females and then release these huge number of sterilized sex, sorted males in the local popul.

Then these males will be in the local population. They'll search and mate with wild type females. Those females will ax, but X do not hash cause males were. And, and essentially, if you do that at mass, you can [00:08:00] suppress population at the local level. And that's how this technology was used to essentially eradicate new world FLS in the us, and then down to the border, us, which agreement with Mexico.

And then they start releasing these sterilized males in Mexico. And then essentially. Move that border, where they suppress them down through all central America and still nowadays every week around. 12 million sterilized P P will be spread over tropical forest on the border with colo and Panama to prevent any reintroduction with these school, one flies back into Northern 

Kevin Folta: America.

Well, I didn't know that that's really interesting. So every week they're releasing irradiated screwworm larvae to essentially block the movement of the. Of the normal screw worms back in the north, north America. [00:09:00] 

Nikolay Kandul: Yeah. And they released them at the pupil stage. So that's kind of one biological feature which made SCR bone flies kind of ideal system for techniques because pupil stage at that flight have a like double, double coat.

It's very robust. And then essentially they just draw for spy in the forest. Then they hatch as a males and all male do is to search for females. Another big, interesting thing feature about that species, that females flies, they made only once in a lifetime. So that means if a wild type Virgin female will made with sterilized male, that female will become sterilized for a life.

Kevin Folta: Yeah, that's a really important part of the technology. Can you talk a little bit about the other species where the sterile insect technique has been done using molecular suppression of fertility? So I, you know, we have other episodes that have been recorded. People can go back in the archives, [00:10:00] but in general, this has been done using different molecular techniques, not necessarily gene editing.

And can you just touch on that briefly? Like, you know, what else has been. 

Nikolay Kandul: I mean, essentially you can, you can sterilize males also with some chemicals that was also down, back in seventies. And then there is also some other system genetic system where you overexpress the recycling response element.

That over expression will essentially mess up translational activation of multiple proteins and then organism can die. And then if you insert female specific into coding sequence of his gene, that's what was max Scott from North Carolina state university. They also develop this system for sported winters.

So and then you have a system where you can maintain your line. Feeding Tetra cycling in the lab. And then once you stop feeding, Tetra cycling, the developing. P like X will develop into the [00:11:00] males. Males will be normal, but all females will die during development. And then you can also release those males into the environment and they will kind of propagate their gene, but their gene will only affect females.

And that's one way to do it. And similar, similar system was used also by OxyTech for a second generation. Of friendly males, which OxyTech now releasing in Florida, where they, again, release transgenic males, males are fertil. They pass these genes into the next generation and then that next generation, every female, which will inherit their gene one or two CORs will die during development.

Kevin Folta: Yeah. So it's really an intricate process. It's taken on a couple of different forms. But the basic idea is to induce some sort of lethality that can be passed on that can be turned off in the laboratory. So at the pupil stage or at the the larval stage, you can turn off this lethality using Tetra [00:12:00] recycling, but then when you release them into the environment, They go out and, and mate and reproduce, but then future generations are not viable.

And so this is a really effective way to do this, but the way that your laboratory and your group, the folks in your group have done this is a little bit different. So can you begin to describe how is using gene editing, going to make this process a little bit different, maybe more. 

Nikolay Kandul: Yeah. And I should say it's not so much gene editing.

It's more gene destruction or gene neurogenesis. So essentially we, we scientists. So we were very excited about CRISPR technology and we were even more excited than CRISPR technology was suggested as a way to create dream drives where. Essentially gene can propagate itself. And if you add additional gene which will piggyback in these self propagating genes, you can kind of change wild type populations of insect, [00:13:00] maybe make mosquitoes resistant to diseases, which wild type mosquitoes generally transmit.

And we've been. Kind of teasing about this system. And I have a background in speciation and relatively soon I realize that the gene drives are kind of a prone to failure, at least right now because it's they have to walk against natural selection. Gene drives how they spread. They essentially cut a specific sequence and then they have to insert themselves based on the Hom.

And then they're very effective at cutting, but they're not very effective at inserting because mistakes will happen. Sometimes these cut regions will be patched. By itself with, and then will be changed and then it become resistant to future cutting. And that's how you create these resistant SOS. And we've been playing a lot with this system, and then we just realized that we really don't need to make gene drives.

We can essentially separate cut slide into one [00:14:00] line. Which now we can propagate because Caline by itself is just molecular scissors, but it's not active without guide, which will guide these scissors to a specific location. And then in the other line will express multiple guides. And again, that line can be brand separately and then home is ghost, but then we have these two lines.

We cross them together and then we have progeny and then the progeny, if then will come into contact with Linell become active. And if we strategically target genes, like at least two genes, one of gene have to be important for female viability. So that gene is targeted, essentially it's destroyed. It's cut.

And then females will die or in some way be selected during the development. And then second gene would be important only for male fertility. And in our case, it's a. Better two balloon gene important for [00:15:00] sperm ation. So essentially doing development. Again, all the males will be sterilized and that's how we came up with the idea that we can have precision guided ster technique, precision guided, because we essentially.

Destroying on knocking down two genes, very highly, specifically, one important for female viability, rather important for male fat and these kind knockout happens during the entire development. Then these F one ag development adult, and then the end product adult would be only male and it'll be completely.

Kevin Folta: Okay. So let me just make sure I have this right. You have one line of, of Drosophila that are containing the CA nine. So the enzyme that does the cutting. So you have the scissors in one and the other one, you tell the scissors where to cut. So either one by itself doesn't have. Phenotype, both of them on their own are simply [00:16:00] carrying the enzyme or the, the instructions on where it should cut.

And when you bring them together in the next generation, now you have the molecular scissors plus instructions on where to cut the genome. And it's targeted towards specific genes that are important for development or. Fity or, or I guess you'd say fertility. So, so fertility in males and lethality in females.

So you have no females that can reproduce and males that now can go out and occupy the fertile females of a population and ensure that they don't mate, against fertile males. Is that. Yes, . I just wanna make sure I have it before we move forward, because it, it seems like a really neat approach that takes the normal ster insect technique, you know, one or two steps further, which, which is really good.

And do you, have you seen some good evidence that when you use this kind of technique [00:17:00] that you strongly suppress a native popul? 

Nikolay Kandul: We have, we have some lab studies where we're tested. First of all maybe I should step down and explain why sterile technique. Walked for some species, it didn't work for another species.

And one reason because you're using radiation right. And then you sterilizing males, but you're also affecting fitness of those males. So then you release these kind of not feed males. They're not really competing very well with wild type males for wild type females. So that's why you have to add them at a huge Ratios like 10 sterilized nails at the minimum to one wild time.

Male. The second problem. It's especially about mosquitoes. Mosquitoes, for example, if you wanna use ster and for mosquitoes, they're very fragile. You essentially can sterilize them, so sort them, but then you still have to release them somewhere and you, it means you have to build these huge factories, [00:18:00] right way you plan to release the nails because you could not ship them far away.

They essentially very fragile at the, at adult stage, surprisingly so, and it's very costly also to separate females. You have to remove them because if you don't separate them, males essentially will be mat with females and they will not be so kind of eager to mate with wild side females and the efficiency of your suppression will decrease.

We made our system. The whole promise was that we knocking out two genes extremely specifically, and we expect that, that our males will be extremely. And we build this system as proof of principle instream. And the biggest surprise was that our PGS a T trans male gastro males were leaving 60% longer than wildlife males.

Exactly the same genetic the ground. So it's quite costly. forSo male to develop sperm. So, but [00:19:00] that also shows that they're very feet and we've also done some competition. Asay where our males were competing with wild type males for mating were females. They were very competitive. And then to also build this system in ADC, GTA and yellow fever mosquito, and essentially confirm that our, again, our males, in case of mosquitoes, they were leaving.

Exactly. They have the same longevity, they have all the same fitness and they also very competitive. So that's, that's one huge benefit of precision ster technique. Some other benefits. They are more SP specific than we developed this system. We actually had our eyes for mosquitoes specifically for aide because that invasive species it's invasive because Desiccated eggs of ADC can hatch one or five years later.

So that's how it's spread everywhere. But what it means for us, for us, it means if we can have these lines in AA, we cross them [00:20:00] in the lab in California, we generate huge number of eggs because we have, plus we sorting parents for the cross, but then every female will lay 200 or 400 X. So we're making.

At least 100 X more kind of solution to deploy in a wild side. Plus in the, in mosquitoes, we can move these eggs logistically anywhere in the world. That basically was our motivation for, for precision guide, still technique. And that's kind of probably the main major benefit of precision guided steel technique.

Kevin Folta: Yeah. So we're talking with Dr. Olai condo. He's a scientist at the university of California, San Diego. And we're talking about new breakthroughs in sterile insect technique. Namely using gene editing as a disruptive measure to sterilize insects that are invasive and problems in agriculture. So this is a talking biotech podcast [00:21:00] and we'll be back in just a moment.

Now we're back on the talking biotech podcast. We're speaking with Nielly condo. He's a project scientist at the university of California, San Diego. Who's been working with precision guided, sterile insect technique. And we, in the previous segment, we talked about what this technique is and what are some of its advantages over traditional, sterile insect technique.

I guess we do have to really think about what are the risks and what are the risks of this kind of innovation in introducing it into a wild population. 

Nikolay Kandul: To say very shortly. I mean, essentially there is no risk. If all the quality control of over process is done correctly because our CA nine line is homozygous guide line homozygous.

All the progeny will be trans heterozygous. They'll have one copy of nine and, and one copy of multiple guide. But if they have it, so all the [00:22:00] females will die during development and all the males will be completely sterilized. So essentially what we are releasing into the environment could not self propagate.

It's a dead end, although it's technically referred as genetically modified organism, but it's not, it cannot self sustain. It's a, it's a dead end. It's only purpose to find and made with female. Meaning with multiple females and, and that's it, but it could not pass any genes and 

Kevin Folta: your, so this is only the F ones that are released, then not the parents that carry just either CA nine or the guide RNA, just the F one.

O F ones. Yeah. Yeah. Okay. So that makes sense now because DF one S only can produce a sterile male that can potentially take up the space on many females. So that that's a pretty good one. Plus this thing is invasive and it doesn't belong here. So that was my next question is, you know, does it have a role in ecology?

Is it food for bats or something that it has some sort of ecological [00:23:00] role. Should be preserved, but I didn't realize that really, this was a very recent introduction that probably doesn't have a whole lot of important role in ecology other than being a farm pest. 

Nikolay Kandul: I would say so, I mean, of course some crickets, maybe dragonflies will be happy to eat.

Japhia but there are many other plenty of Japhia which can sustain population of crickets or dragonfly, or mosquitoes probably will be preferred dragon for dragonflies Soso. Suzuki's a very recent invasive past. 

Kevin Folta: And you mentioned some other Insects that were being engineered with this kind of precision guided sterile insect technique.

What about the solids associated with citrus screening? Is that something on the radar? 

Nikolay Kandul: Yeah, we're still, I mean, we, we're thinking about it. In fact, in the lab of Omar ABAR, who's a food professor at UC San Diego. We are trying to establish transgenesis of seal. Once we establish [00:24:00] transgenesis, we can very easily port this technology because precision guided still technique.

It's a, essentially it's a platform it's only methodology. If we can make transgenic insects in the one particular species. We know that Caine will most likely walk because Caine walks in bacteria and in humans in all different organism where it was tested and the same goes about GNA. So it's just methodology it's once we establish transgenesis, we can build PG S a T.

Kevin Folta: And you had mentioned that, you know, this is CAS nine guide RNAs. How hard is it to go through a regulatory process to be able to introduce these technologies into any given scenario, because we really need this for the Asian citrus. SI, we need this for the Ambrosia beetle. We need this for a whole bunch of insects that are.

Vectors of agricultural disease. And is this something that could potentially be deployed very quickly? 

Nikolay Kandul: We hope so. Agri [00:25:00] it's one company very licensed with a patent Precision guided steroid group patent. And they're actually now running S D a tests of targeting DSO Suzuki in, in Oregon in the, in the greenhouses as a past for blueberries.

So they're already testing this system in DSO Suzuki. We also, as I mentioned, we also build this system in a C GIDE and we're very keen to start deploying it. And we. Preparing application to EPA because mosquitoes are regulated as bio pesticide and they belong to the EPA any kind agricultural regulated by us department of agriculture.

And that's also under, under that umbrella. But we're going that route. We wanna apply it as soon as possible because it it's, it's scientifically very attractive idea. It works in the lab. We wanna test it in them 

Kevin Folta: in, in actual environment. Oh, very good. And I know that this work never happens in a [00:26:00] vacuum who are some of the collaborators in the work?

And you mentioned Dr. ABAR who are some of the other folks who were involved in making sure this could. 

Nikolay Kandul: Max Scott who is the professor of North Carolina state university at Raleigh. And he also built a system, the other system to recycling response element system for Zo fellow Suzuki.

So his lab was working also very hard and they're collaborating with us on. P G S a T. We also have collaborators from university of California, Berkeley, who in the lab of John Marshall. They help us to model scenarios and to essentially use mathematics, to figure out which currently available technology would be the most effective for suppression of.

Kevin Folta: Oh, really good. And is there any hints as to when this kind of thing may be available to the farmer or for widespread deployment? Is there any discussion of a timeframe. 

Nikolay Kandul: It is a discussion. We, we, we got funding from a [00:27:00] California cherry board to develop PPGs T in Zo, Suzuki, and, and again, agen right now is doing some environmental test, the face technology.

And once they do that test, they probably have to do test another one at the higher scale. And then they'll have to submit. Data showing the efficacy, these methods to us department of agriculture. And again, I also mentioned that we have this system in mosquitoes and we ourselves also founded the company called VEC.

And we're also pushing very hard trying to bring these technology into the field. And we are preparing experimental use permit for the EPA to test this technology. But my understanding. Ox attack was pioneering, these genetically engineering solutions for mosquito control. And we're trying to go through laboratory nightmare in us over 15 years.

And now they [00:28:00] established the essentially regulatory path. And now we are following that path, our technology, and we hope once we do. Field trials, we'll gather our data. Then we can submit data efficacy data back to EPA. And after that EPA satisfied, they can essentially register our products. And then after that, we'll have to go to.

Different states at the individual state level. Also get some regulatory approval and then, and farmers can get access to it, but it's a relatively long process. The good thing. Now that process was established and primarily by OxyTech. Yeah, 

Kevin Folta: OxyTech did a great job with getting through it. And they were excellent with their public facing side in terms of discussing what this technology is and how it can be beneficial, but still there's so much pushback that's there.

That the only way we're going to get through it is to get people who listen to this podcast to really work for [00:29:00] us. And, and, and when I say work for us, I'm saying work for the farmer and work for the broader scientific good. Discussing this topic, write blogs, you know, get in social media and talk about the good things that can come from this kind of technology.

Because to be honest, we really need it yesterday. And especially for the citrus SIL, that would be revolutionary. If we could crush the levels of that thing. It's the vector of the citrus screening disease. That's destroying citrus industry in Florida and present now in California. So, this is a really urgent thing to be able to integrate these new technologies.

So if people wanted to learn more about this, is there a place that you could direct them online, like to a website or social media presence? 

Nikolay Kandul: I mean they can go and check Omar via lab webpage at UC San Diego. Max Scott lab webpage at North Carolina state universities. Essentially they can also Google my name or Omar via and C publications.

There is also Wikipedia page about Jaso [00:30:00] Suzuki, which described relatively up to date information about these pest and, and some advances. Coping with it. 

Kevin Folta: No, very good. Well, thank you so much for your time today, Dr. Condo, when you have your next big breakthroughs, could you please be sure to let me know and let's talk about it here, because this is an exciting twist on an old technique that really worked well before that now has been given the precision guiding of modern molecular biology.

So thank you very much for joining. Thank you so much, Kevin, and as always, thank you for listening to the talking biotech podcast. This is another amazing breakthrough that you should be sharing. Share it in social media. Talk to your friends about it. Talk about the ways in which we can limit the, the impact of destructive insects that don't belong here in the first place.

These are introduced in invasive species that cause tremendous damage in the fruit and vegetable industries. And now we have technologies that can eliminate. [00:31:00] With very little risk and actually decreasing the use of pesticides and other impacts on the environment. So thank you very much for listening to the talking biotech podcast.

And we'll talk to you again next week.