 Nico is a professor in plant physiology at the Scuola Santana in Pisa, of which he was in the past also the director, and he will talk to us about innovation through DNA and RNA technologies. Thank you for your new income. Thank you. Thank you very much for the invitation. I'm glad to be here. And as soon as my slides will be on, I will try to be fast because I realized that we are already late. The good news is that I tend to be fast. The bad news is that I have a lot of slides. So let's see which will be the final result. Okay, so I'll skip the first one because this is just the title, and I will move to the obvious. We already know all this, the world population. I'm trying to get this working, but no, no, I'm trying to have a pointer. Ah, okay, okay, okay. Okay, okay, okay. Thank you. Thank you. Okay, so the world population is is is increasing and we are heading towards the rewarding numbers in terms of world population and you see around 10 billion people in 2100. Okay, we will not be here probably, but it will be a problem for those who will be here. This will lead to the need for more food. There is no doubt about this. There are a number of estimates online. So I don't know how reliable are all these estimates. But some of these say that we need 70% increase in food production, which is which is very worrying because it's a lot. Okay. So we need for sure to change something in the way we produce food. And the other important point is that we know that all this is happening in the context of climate change. So not only the world population is increasing, and we will need more food, but we will need to produce this food in a context which will be more difficult that in the, than in the past. We will have less water in some places more water in other places, higher temperatures. So we will need to change a lot in the way we do agriculture and reproduce food. So we will need to choose among different scenarios. We have we have to do some something of course we cannot stand and see, but we need to choose among different scenarios and I found an interesting publication which came out very recently which is the result of a European project, which presents four different scenarios. One is the plant innovation scenario. The second one is your food health choice, the food emergency and the reject tech, we will see rapidly now, which each of these scenarios propose. One is financial means growth and tech technological options unlimited so what we can use all the available technologies we can, we have unlimited the financial resources to reach the goals the scientific goals, the innovation goals to produce new technologies to innovate at the fastest possible speed. The second scenario is is is what Giovanni carada described us is the consumer driven market. And when I say consumer driven market I do not only mean something which is of interest of the supermarkets because the consumer will decide what they will sell, but the consumer decision. What influences the policy makers and the policy makers will influence science in the end, and therefore will impact the in the direction that research will be taken will take. We have a food emergency scenario, which is something that to me reminded the COVID emergency. Okay, so we had a health health emergency where we have a pandemic situation worldwide, and science was alloyed to go faster. Because we needed the vaccines, we need therapies. We need monoclonal antibodies, we needed the RNA based vaccines which were never used before, and all this happened very fast. So that scenario demonstrates that it is possible to face a specific emergency route. Here's an example by applying an emergency scenario in which we will provide unlimited resources, but which are focused to solve an emergency. I don't consider this good because we, we, unless the emergency is a real emergency something which cannot be predicted. I just showed you that we can predict that the population will increase, that the climate will change, so it's not really a scenario which we can apply to what we know. We have something that again was described by by Giovanni, which is the reject tech scenario. So, there is a general mistrust in science policy makers and the agri-food system, and we want agriculture to get back to the past. We don't want innovation or we don't want anything which is new, and we want to keep things that not as they are, but even to go back to the past. So technology is bad. We have to reshape agriculture, but based on ecosystems and needs, et cetera, et cetera, not using technology. So the big difference in this four scenarios is this. So the fact that all the technologies that involve DNA modification are not desired or not accepted by many of these scenarios. So what makes the real difference between these scenarios is whether we can, we will, we will be able to use or we will be hampered to use technologies that influence DNA. And this leads to the first part of my talk, which is DNA technologies, so which technologies are available to modify DNA so that we can produce more, better food or, you know, we're using a sentence which is very popular. We to produce food in a more sustainable way. So, and this leads to a new genome techniques as they are defined, which are exactly the technologies described by Giovanni, so the new technologies of DNA manipulation. I'm very worried when I use these words because DNA manipulation is not fantastic. Okay, but I know this is the way it is used generally. And in order to identify what they are, I show you this, sorry, these two covers of science and nature, they date back some years, and they presented the technology of CRISPR-Cas9 as the breakthrough of a year. So we are not, when we talk about something new in science, we do not necessarily talk about something that happened yesterday. It might be a technology, a science advancement that happened five, six, 10 years ago, but that is now ready, the technology is ready to be used in agriculture to produce new food. But if we have new genome technologies, there should be all genome technologies. Okay, it is nonsense to say that we have something new if we don't have anything old. So let's see what they are, the old genome technologies. But, well, this was already said by many of you, also in the questions. So, breeding, human beings have been improving crops since millennia. And this was done by modifying genomes, because if you cross two species, what you will end up is a mixed genome of it over two parents. So it's a big, this has a, breeding has a huge impact on the genome, which is an impact which is even stronger than transgenesis. So if we speak about which is the impact, the actual modification on a genome by simply talking about breeding, breeding, inevitably has the biggest impact, because it's two genomes, possibly of two relatively unrelated species, but which were compatible or made compatible by the breeder that are mixed together. So we have mutations, plants have been improved by mutations since centuries. And one way to mutate a genome is to use radiations, which will impact on DNA at random. It will modify at random the genome will lead to 100 of the negative effects and possibly to a few positive effects. In this slide, you will see the radiation field, which was in near Rome at Vienna, where a source of radiation was in the middle of the field, and all the crops grown around where irradiated to get to increase variability could be picked up evolution in a way in a very strong way. Okay, harsh way. And this, this, this approach led to many, many crops which are which are actually used and consumed. One example is the pink grapefruit, which is obtained which was obtained by by mutating the genome by radiate by using radiations. And you see that this Ruby grapefruit obtained by radiations is sold as a jam, which is organic. So we have, we have a paradox here, in which we have two scenarios together on one side we have the your food health choice which is for example, which is organic. And on the other side, the radiations used to produce the crop that was used to produce this jam. So it's a, it's an example of how these scenarios might be in conflict, and the consumer might not always realize about this conflict. This is genesis course GMOs GMOs are perceived as a kind of failure. Okay, especially in Italy. Okay. Well, GMOs we don't have them. We survive we had no problems. We forget that we import GMOs from Brazil from other countries, and they are not really zero kilometers crops if we import them from Brazil. And this is the surface of the actors, which is currently grown with GMOs. And, well, saying around 200 million actors that does not tell you much. Okay, how big is the surface. Okay, well, let's compare it to the surface of Italy. So this is the wall surface of Italy, which is around 30 actors, million actors of course. So the surface which is cultivated with GMOs is huge. Okay, and we are talking about corn, soybean and cotton. No, no, no, no more. So GMOs were a great success technology which had a great success and a success which Italy did not take any advantage of, because we are paying the costs of this technology by having to import from other countries that GMOs we are not allowed to cultivate. And this is the scenario. Okay, so here I put together a number of claims that are used to convince the consumer that that product is better. Because it's GMO free, because it's a non GMO, a natural product, etc, etc, etc, etc. So this leads to the new genome techniques so with this part I, I hope I convinced you that human beings have modified genomes since ever. So we are not talking about now these crazy scientists are starting to modify the genomes with something new is simply that we take the technology is going ahead. So, and we are talking about gene editing. And which is the, the characteristics we wish for gene editing to be safe, precise and predictable, because not even breeding is inherently safe, precise and predictable. So we are talking about more, we cross to plants and we hope that the new plant will be better, will be safer and and and showing the traits that we want. Okay, the reality is that we cross and we cross and we cross fingers as well, because we hope to get the desire result because it is not true that you are doing genetic engineering, because you have a project and then you realize the project is based on trial and errors, the transgenesis process. So we need something better. Okay. So what is genome editing and what is CRISPR, because the technology which uses genome editing is called CRISPR. So it's something that was discovered by a manual sharpened engine if I don't know, which we're working on on a bacteria, a useless bacteria, where we're studying an apparently useless process biological process in an apparently useless bacteria, and they discovered by chance. There is a mechanism that is happening in that bacteria, but that can be translated into a very powerful technology. And for this discovery, they got the Nobel Prize a couple of years ago. So the technology is based on this protein, which is cast nine, I will not go to the detail but you can recognize here a double elix of DNA. So this is how the system works. This protein will cut DNA and will be directed to a precise position by the RNA sequence so we can use RNA as a sequence we will find the complementarity in DNA and will then act at that specific position. So this will allow what is a site specific in a way we can say mutagenesis or editing of DNA. Well, CRISPR means this I don't even try to read it. Okay, so it's a it's a forget it. Okay, you know that it means something about you will never be able to remember better CRISPR means clustered regularly interspaced the short palindromic repeats. It means that this is not very friendly terms of communication. Okay. Cas9 is easier. It is a CRISPR associated protein number nine. Okay, so just just because acronyms are puzzling. I wanted to show you that there is something behind the name, but is is something that you can you can forget you know that it happens. Well, Cas9 is a molecular system. So it cuts DNA, but it does not cut DNA in any places it cuts DNA exactly in a position. It cuts DNA of course, and it cuts DNA following an RNA guide sequence so you get a site specific DNA cut. This site specific DNA cut is followed by a repair of DNA, which might modify DNA in that specific position so you will, you will get variants. You may get a knockout. If you choose the right position so you switch off the gene, or you can modify the gene, you can get the gene to be more similar to a wild relative. And this is how it works. Very simply Cas9 is this light blue molecule. It opens DNA. It uses, which is the orange double elix. It utilizes the purple RNA guide. You can design it in the lab. And this RNA guide is used to define the exact position where DNA will be cut and then repaired. So it's simple and efficient. Okay, in my lab we started using it and it worked the first time we tried. It did work. It is faster, more efficient than transgenesis. Great. So this technology can be used to modify plants and to modify plants in specific traits because now we know thanks to the genome projects we know the genome exact sequence of the genome of many crops. So we can identify the genes which are responsible for the specific traits, which is still difficult. It's not an easy job. And we can modify them for getting better food and better agriculture. One of the very first CRISPR, which is not a plant, which is a fungi, which was produced is this mushroom which resists browning. And immediately it led to this. So, and this allows us to say that, yes, we are using a new genome technology, but the perception is simply that is a new generation of GMOs. And with all the easy terminology that can be used by Greenpeace and all these organizations which are against, which are for the reject tech scenario. Because this is exactly the reject tech scenario. So we have a new technology, but if we adopt the scenario number four, which is reject tech, it will stay in the labs and will never reach the supermarkets. And it will never reach the developing countries. It will never reach any place where it can be used to solve food related problems. Now, many companies predicted that we could have CRISPR plants in our dinner plates in five years. Usually, it's always better not to make predictions because usually you fail. Okay, but in this case, it was not too bad. So we should have had the first CRISPR dinner in 2020. But we now have the first CRISPR modified crop, which is on the market in Japan. So there is at least one CRISPR based product in Japan. And this is interesting because Japan was one of the country which was against GMOs. People in Japan didn't like GMOs and they were no GMOs in Japan apart from some blue colored flowers which were initially produced in Japan. And the second point which is of interest is that this is improved in nutritional value because it's a rich thing, GABA, which is supposed to lower your blood pressure. Okay, so it's a nutraceutical product which is aimed, if you consume it, to improve your health. Without taking pills, you eat tomatoes. This is the oversimplified scenario. And this is more recent, well, 14 February is very recent, is wheat which produces less acrylamide. You will say, why should wheat produce acrylamide? Well, all food that produces contains good things, but it might contains also bad things. Okay, so usually bad things are here and good things are here and bad things are here, but are still there. So we want to reduce the amount of acrylamide which can be produced by wheat and this was obtained by genome editing demonstrating that we can use this technology to produce better food. Of course, we will need also more food and of course plant more resilient, etc, etc, etc, but this is feasible. The European Commission is discussing how to regulate these new technologies. We have two scenarios on the table. The plant innovation scenario of the reject tech, who will win, we will see. Politics will decide actually and then the consumers will be important in this decision and this will set the direction that Europe is taking for the use of this technology. Remember that Europe is not the world. Okay, that is China, that is the US, that is South America is Africa, so there are other continents that might take different decisions than Europe, and we will see. The point is that CRISPR-Cas9 leads to permanent genome modifications which are different from transgenesis. We are not talking about inserting foreign genes inside the genome, but they are permanent genome modifications and this might be one of the reasons why we have a very jet tech approach and perceptions. Okay, it was said there is also something religious, so God created that DNA, why, how they are modifying it. So the permanent genome modification is perceived as possibly dangerous scientists playing God, etc, etc, etc. So let's go to RNA technologies, which are something newer and which are different from DNA technologies, although CRISPR-Cas9 in a way is partially an RNA technology because it utilizes RNA as a guide to cut DNA. So let's remember that RNA is the first product of gene expression, which will lead then to a protein and finally to phenotype. Okay, so if we are talking about RNA technologies, it means that we are going to modify the way RNA is present in the cell, so we are not touching DNA, we are playing with this molecule. The DNA, the genome isn't touched, but we can act downstream. So why should we modify the genome if we can modify the expression of a genome and more specifically the expression of a specific gene. In this case we can get a transient modulation of gene expression. We'll steal this be rejected by people who reject technology, so we do not modify the genome, we modify the way the genome is expressed. Okay, and the genome, the way the genome is modified in its expression happens every day. We have sun, the genome is expressed in one way, if it rains, the genome is expressed in another way, so we are simply exploiting the ability of the genome to adapt to something. And we can do it because there is one process which is very important, which is the RNA process, which means RNA interference. Again, scientists are not very good in finding terms. Okay, because if I tell by this new tomatoes produced by RNA interference, it doesn't sound so it is not friendly. Okay, the word interference at least. But the concept is that you can have a sequence of smaller and a smaller and a can enter the cell and will recognize a sequence of RNA and will destroy it. To make it simple. Okay, so you can use a small RNA sequences to destroy messenger RNA so to destroy the message messenger in it so you can you will block the expression of that gene, or you will strongly reduce the expression of that gene. This can be done. This can be done to to to kill insects, for example, if you have a plant, which contains this small RNA, which encodes a gene or better encodes a sequence for an RNA in the insect, which is an housekeeping. So producing a protein which is essential for the life of the insect, then you will kill the insect. Okay, because the RNA will down regulate the expression of one gene, which is essential for survival. Of course, you can design RNA for that specific insect. So only if that specific insect, having that specific sequence of RNA is targeted, then it will be the only one to be killed bees will not be affected. So for example, you want to kill a bug, you see you you design an RNA which will kill that bug because it will recognize a sequence of RNA, which is specific of that bug, but completely absent in bees. You can treat and the insect will eat that sequence and simply by eating it works. So the insert eats this small RNA sequence and it is killed because inside the cells, the process of our interference will block the RNA and RNA, which is essential for life. Okay. So by spraying. So you simply spray the plant with this double stranded RNA, it will be uptaken by belief, and depending on the sequence of RNA you utilize you can kill some insects, fungal pathogens, viruses. And it has been demonstrated that you can block viruses because you can target RNAs which are viruses, many are many viruses implants are RNA based chewing insects, etc, etc, etc. RNA technologies, which are most promising are based on double stranded RNA foliar spray. So it's not, we are no more talking about modifying plants, we are not in the city industry producing a new variety of plants, obtained by gene editing or by disease or by breeding, but we are talking about something which will be exploited by companies usually selling fertilizers pesticides because it's an RNA based pesticide in this case. But we can, and it has been demonstrated that this happens in a trans kingdom way so the RNAs of the plant can interfere with the fungal RNA, but also the fungal RNA can interfere with the plant RNA so you can interfere with this mechanism in a way that the plant wins the battle with the pathogen. And even more recently my lab demonstrated that this is also possible in nature. We have a plant which is over expressing one specific small RNA. It will be released in the soil, and it will be uptaken by a nearby wild type plant, and you will modify the way the genes are expressed in the target plant by simply growing it nearby a plant which is producing that specific micro RNA. You can simply add a synthetic micro RNA which is identical to the natural one to the soil, and you will modify the expression of the gene in the receiving plant. And even that micro RNAs are important for a number of traits which are important in agriculture from stress biotic and a biotic stress develop development, etc, etc. You can utilize an RNA spray to modify plant development to modify plant resistance to stresses to modify plant resistant to insects. The RNA spray for transit modulation of gene expression is a new technology that might be more perceived as more friendly RNA is non toxic. Of course, we eat RNA every day we will have RNA for lunch. I'm pretty sure we will have also DNA for lunch. And we eat normally so it is not supposed to be dangerous for for health, and it will be transient. It will be like any other agrochemical product which is preyed on plants, but it will be more specific. And it will act on specific insects specific traits, and it will be able once developed to improve the plant productivity and food production. There is not one single technology that will save the world. We will need an array of technologies and many of them were mentioned that we need precision agriculture we need artificial intelligence. We need the DNA and RNA technologies we need better pesticides. We need a better farm management technologies, etc, etc. But if we use all these technologies, we will able to save the world and the human beings will be still here in a heavily populated world, but possibly living well and healthy and possibly more than 100 years in good health. Thank you very much for your attention. Thanks a lot periodomenica for the very interesting talk. We have time for a couple of questions online. Thank you for the great talk. I have a question concerning the Chris mechanism. So when say you want to change the color of that mushroom, so that he doesn't get brown if I remember correctly. We know that you can associate this property with a specific site that you target with RNA. But I mean, once you cut I have two questions. The first one is, can you also manipulate the way it's reformed in that specific place. And the second one is, how do you know that the specific site doesn't correlate also with other properties that you didn't want to change. I have answered it to the last question. You need to know very well the process. Okay, so the genes involved, of course the gene involved in calls for enzyme which will oxidize one compound which will become brown and so you want to kill that gene. You can do it by the knockout approach of CRISPR. So one approach of CRISPR is simply you target the gene to make a version of the gene which is no more functioning. Okay, it's the easiest way of using CRISPR to get the knockout gene. You can also do what you proposed, so you can repair the DNA by either hoping that it will be a new sequence will be in the repair process. Okay, a sequence will be slightly changed in a way that the protein is more efficient. So in that case we will have a mushroom that get more brown okay undesired about just to explain what what happens, or you can use a guide to, you can even use the guide to insert a new trait, a new piece of DNA, but in that case, the technique is used to make a GMO. Okay, so I'm not talking about that. The problem of GMOs that one problem GMOs is that you cannot predict the site, the site of insertion of the trans gene, the foreign gene you are inserting the genome. You can insert the foreign gene in a precise site, but in that case, it's a GMO. Okay, so it's not the new genome technology I'm talking about. The new genome technology I'm talking about is a technology that does not leave anything in the plant that is or can be called as transgenic. And this has an impact on regulatory regulatory aspect, while you can trace any transgenic plant that very easily with this technology, you will not be able to trace it. Okay, so if the country. I think that the country of your preference will start producing seeds modified with this technology, and we'll start selling the seeds to us. And no one explains how they were obtained. There is no way we can discover it. And we will use them. Okay, so, and you know that to enforce any law, you need a method to detect the people that are not respecting the law. And in the case of Cas9, it's reasonably easy to hide the modification, it could have been happened by chance in nature as in a laboratory by using CRISPR. And this has a big impact in terms of the market. Okay, because a country which is not regulating this process carefully can very easily have a variety that are superior. While we are discussing about, should we do it? Should we, how do we regulate it? We might start receiving this plant. There is a nice story about an orange petunia which was on sale in Italy for a while. It was transgenic, it was just by chance entered by error, and people were very happy because usually petunias are not orange. And we're buying them and then someone discovered that were a transgenic plant that were not illegally, it was by error probably imported in Italy. So if you don't have a method to detect the modification, it's very easy to alter the market. Okay, thanks to like everything is very clear. I just have one remaining curiosity, which is, I mean, once you use CRISPR as saying the easiest way that you say it, so you just remove that, you deactivate or remove that gene. I mean, I'm not an expert in genetics, but one gene is coding for more than one property, I guess, right? Well, I'm not a geneticist. One gene is encoding. The dogma is one gene, one protein. We know that this is not true. Okay, there is one gene, and there are, and there is the possibility that that gene produces different variants of that protein. Okay, but usually, let's say usually one gene encodes for one train. Okay. Okay, no, I just want to reiterate it like in the most naive and simplest way possible, my question. So isn't there the risk that once you change the color of the mushroom without being knowledgeable about that you're also changing other properties that you don't want to change and you make it poisonous. Yeah, it is possible. It's the same when you cross two plant species. You cross two plant species and you mix two genomes. Okay, you don't know how the interaction of the two genomes together will be expressed into a new plant. So you might, and there are examples of breeding leading to toxic, for example, to a higher accumulation of alkaloids in some plants. So it is not the risk is inherent in any technology. Okay, but it's not, but it's not the new technologies which are also the old technologies are even more dangerous. What you need to do, of course, is to evaluate carefully the final result you get something which is not done with breeding. Okay, so my lab produced a tomato which is black. Okay, it is on the market. No one asked us to prove that it is not toxic. As far as I know, nobody died. Okay, but this is the only toxicological result I can, I can, I can discuss with you. So no one claimed that relative died after eating our black tomato. So it's nonsense that on one side that you have to spend two million euros to prove to carry out toxicological test on GMOs or any modified plants. And on the other side, you can cross anything, obtain anything weird. Okay, because a black tomato is weird. Okay, and that's the reason why they're selling it. It's weird. People like it. But in theory, you should check. Okay, it's the same. We don't have time. Sorry, but it's we are very short. No way. Otherwise, we cannot have lunch. Choose between question or lunch, because we have to be here are too sharp because the speaker or two has to start at two so sorry about that. Anyway, I mean, we'll be here all day. So just in case you need to ask questions. Sorry to have to interrupt, but we are very late. So our last presentation of the morning is from a speaker who will be connected online. Leo Violini who