 So thank you all for coming to this presentation, the 2023 Junim Editing Year in Review. I present this usually the first or second January every year in the cycle to talk about the exciting things that happened in genome editing. It used to be called the CRISPR talk, but things have actually moved beyond that, beyond CRISPR. So it's using a little bit more of a general term. So I am Steven Gager. I'm a PhD in molecular biology, and I'm currently a actively working scientist at a company called Corteva AgriSciences. Although here I am in second life as Steven Zewfly. The basic structure of the talk doesn't change very much from year to year in terms of the basic way I structure and organize the different parts. But this particular year, I'm going to go back over to background again, give you some basics of how CRISPR works so that you understand the technology, you can understand some of the therapies that are coming to the consumers or other things that are hitting consumer shelves. We'll also be talking about delivery advances. I think a lot of times when you think about science and pharmaceuticals, there are amazing chemicals, amazing therapies, amazing RNAs, or DNAs, vaccines. But in fact, it's not the science itself that's important, the compound. It's how you actually can get it where it needs to be to be effective. And so I usually spend a good amount of time on that particular topic. Target genes and modified organisms are just kind of that catch-all of interesting genes or interesting modified organisms. One thing that I tend to focus on in these are things that either are relatively both have to be novel enough to be interesting for the year or something that's kind of cute, clever, or I think will be impactful in future years. So I think that's something to keep a mind and eye on as well. I can't talk about everything that's going on in the CRISPR field. There's just too much. But every five days, I get an email, PubMed search list of about 15 different articles every five days. And then new tools. New tools are things that I think, again, similar target genes and modified organisms, these are a novel way of thinking about how CRISPR as the technology works or maybe some new application that's, again, not really in the therapy or consumer space. And finally, some cautionary tales. Again, like any new technology, there are some aspects of it that have to be critically understood and reviewed and make sure there are no consequences to its use. And so with that, I do want to make an opening clarification that I am a researcher at Corteva. It is an ag biotech company that operates within the space and it actually does have quite a bit of intellectual property holdings and interest in the area. However, I mean, I think, unlike some other years, there's actually nothing in particular to talk about for my company in this particular year's talk. Nothing should be construed as an investment advice or a company for looking statements. I'm not here as a representative in any way of the company. OK. So let's talk a little bit about what CRISPR technology is. And what I have up here are some diagrams that show some different variations on the CRISPR technology. What CRISPR, in essence, is a kind of immune system that originally was evolved in bacteria to do this very specialized jobs of basically targeting and eliminating viral infection. Now, like I talked about, I think, two years ago, the actual original form of these proteins actually probably had to do with things called jumping genes. They are actually things that the main reason for being is to find specific sequences of DNA and make cuts. And so what I'm showing here in this diagram are some examples of a Type II effector complex, something known as Cas9. That's the most popular one. Type V complexes, Cas12a is a good example. And what you're seeing in the blue is a bunch of protein. It's a protein complex with RNA. And that RNA is being represented as either purple or black. And that RNA, because of the property of how DNA works, DNA is a double helix, it will bind to and find a target sequence, and that's what guides it to the protein to a spot to make the break. And as an interesting evolutionary side, well, somebody's actually have learned to target RNA. And so that's what you see on the bottom hand side is a Type VI complex. Again, the blue blob is combined with the black RNA. But in this case, it's actually base pairing and targeting that magenta sequence, which is a target RNA sequence. And so in those cases, they may be of all for trying to fend off RNA viruses. But in many cases, these have been adapted to a use in gene regulation of actually targeting a system's RNA to basically turn off a gene. So it's an interesting part of the evolution that these have this basic type of activity, which is a guided endonuclease, cut DNA. But then evolution can make that do lots of different things. So for the purposes of that basic core structure and reducing it down, what you have here is a diagram of DNA. So a blue strand and a red strand. It's double-stranded. And what these will do is make a double-strand break. And the consequences, again, CRISPR doesn't do this. The host factors that the cell responds to that double-strand break by doing a couple of different things. And one of those things is to potentially insert DNA into that if you're providing some template. Or in some cases, I should say in general, it gets repaired by the host repair complex. Sometimes it gets repaired with the mutation. Sometimes if you are pushing the way it tries to repair it, you can get various editing or inserted regions. And sometimes it just goes back to the sequence it was before. And that's not very interesting, but that actually is something to keep in mind that those typically happen. To go through some of the other proteins, I want to go through the fact that, as a reminder, CRISPR was not the first so-called genome editing factor. There were things known as talons, things called mega-nucleases, things called fingers. And these also have a place within the genome editing landscape right now. And I talked a lot more. I focused a little bit more on those last year, because these have properties of not needing a guide RNA. The ability to find a particular sequence of DNA and cut it is intrinsic to the protein by itself. So in some cases, that can be very useful. And then there's another class of proteins known as argonauts. And what argonauts do, they can use RNA to guide them or DNA to guide them to a place, but they don't make a double-strand break. They usually make single-strand breaks and can only target single-strand DNA or RNA. In the first place. So this really kind of covers the general activity and the realm of things and the tools that people work with. And just a reminder that, and that's what's shown here in the top diagram, is just this example of what you have is you have a protein complex with argonauts or CRISPRs and that RNA base pairs and targets specifically to that target red strand, which is, again, typically, we'll talk about that being DNA. And with that DNA, it base pairs and then it cuts it. It cuts that molecule and then releases it. Now, what's kind of important here, and I want to take the time to just highlight this very specifically, is that this base pairing is not always exact. What can happen in cases is that the red sequence does not exactly match the black's complementary sequence in a way that the place where you try to cut something or places away from where you try to cut also get cut. And so that's just an important part and aspect of how we treat this technology. Now, the bottom part of this slide, the diagrams here, are showing some variations on the steam that David Liu, who is most well-known, he's at, I think, Harvard, where they added extra protein packages onto that protein. And by adding some extra activities and packages and also taking away the double-strand break activity of the cast protein, they've turned them into so-called base editors. And the main idea here, as you can see in the diagram, is that this additional cytidine deaminase purple ball, or maybe an adenine deaminase, which is this blue hexagon, that those can interact with the DNA in a way that's only trying to modify one single base pair. And because the protein itself has had its double-strand break activity removed, although it still makes a single-strand nick, then instead of making that dangerous double-strand break that the cell responds to, you usually have this very small editing package change. And then prime editing is an example where by adding on what's called a reverse transcriptase package. And reverse transcriptases are things that take an RNA sequence and then actually synthesize on top of it a DNA sequence that by adding this package onto a base editor, they can now insert small amounts of specific DNA into that target site. And so this is a nice way to modify genes in a prescribed specific fashion where you're not just limited to the repair usually just changing something. You can actually add extra DNA to that sequence. So that is a quick summary of what the basic idea and activities of these types of classes are, the proteins, how they can interact with cells, how they can basically accomplish the role of genome editing. For those of you who need this topic, this was something really only characterized as a tool in the year 2012. And then Dalba and Charpentier won the Nobel Prize in 2021 for this work. So a very quick turnaround time for Nobel Prize, but it's warranted. And I think it's warranted that if you look at the amount of work and the things you might hear in the popular press or the science news media, there's a lot going on in this field. So hopefully I can walk you through some of the most, I think impactful and interesting things from this year. Coming in under the wire for 2023, you might have seen this in the headlines a few weeks ago, right before Christmas time, that for the first time, a government health agency has approved a CRISPR therapy for patients. This is past clinical trial time. This is actual approved therapy that a person can get at a hospital now. So the basics of it, and I just have the text, a crib diversion of the announcement here, as talked about in Nature magazine, it's called KESKEVY. And it's something I've actually highlighted and talked about several times during this year in review. It's a way of trying to treat sickle cell anemia or beta thalassemia, two diseases of hemoglobin that humans experience that have very severe phenotypes. You have the blood can clog up in a way that you get incredible joint pain. In many cases, people do not survive. In the old days without medical intervention really did not survive past their 20s. And the current therapy for this is to give them blood transfusions. So basically on a regular I think two or three week basis they have to get a whole blood transfusion of fresh blood from a donor in order to basically carry oxygen around their body. Now this is a prevalent disease because it helps fight off malaria. So in places like the Mediterranean and Africa you see a high prevalence of this disease in those populations. And so the basic idea behind the therapy is it allows for the expression of gamma hemoglobin which is the fetal hemoglobin. And so when that fetal hemoglobin was turned back on it's a completely different gene in these patients than they can now transport oxygen around their body. And so the main interest here is that this has been under development for a long time. It's one of those easy to envision first targets for CRISPR gene editing. And what the company published is that in a trial for sickle cell where they followed 29 patients long term that one year after the therapy, which is extensive they actually have to have their blood cells taken out of their body, treated, put back in they have to stay in the hospital for a month. They, it's not, it's a bit uncomfortable. But they had, they were relieved of debilitating episodes of pain for at least one year. And then it's 42 patients that have a severe form of beta thalassemia. 39 did not need red blood cell transfusions for at least one year. So I think this is what we saw in those data. The UK was ready and did approve. There were no clear side effects that were worse than the disease. And then basically about a week later the US Department of Health also approved it in America. So this is now the first off the shelf gene therapy treatment for patients. So again, exciting, probably the most exciting thing that happened in the world of CRISPR this year. There's another, so there are a lot of ongoing clinical trials, there are a lot of exciting diseases that people are looking at and trying to treat in regards to using CRISPR for it. This one was particularly interesting this year because they demonstrated the first kind of proof of concept within patients that you can get a long-term reduction of a familial hypercluster emia again, which basically creates extra cholesterol in your blood that can lead to heart attacks other cardiocardial problems. And what's kind of exciting about this one is that it was therapeutically showing a reduction long-term from a single dose of a bodily produced metabolite. And so that's what they were showing us that the low-density lipoprotein C reductions up to 55% at these therapeutic doses up to 84%. And in one case, in the highest dose cohort, a 55 reduction was durable for about 180 days. So this is again, this is one of these diseases that's treated on a very continual basis with quite a bit of drugs, statins that can have their own side effects. So yeah, LDIs is not good. So this was really interesting in terms of showing this like durable type of effect on what is human metabolite that has a disease prevalence to it. So the other thing that's actually really exciting about this one, and this is something to keep in mind is that this type of target therapy that can reduce cholesterol in people with disease, it actually could be also thought of as a prophylactic for healthy individuals. That if you're someone who is dealing with high cholesterol but you don't have a genetic or known clear genetic predisposition to that, this also could be a prophylactic for your health as a normal person to fight off and wore off cholesterol for whatever reason. So it's interesting, I think that's been interesting. Well, again, exciting for therapy is I think what I'm really trying to say. All right, so in terms of stuff that's coming to consumers, what we have on the store shelves, although I think the main retail chain is gonna be through restaurants, at least initially, is the first edited plant that we eat. So this is something introduced by pairwise. They now have their own CRISPR edited plant brand name known as conscious greens. And so I have a news release here from seed world but also some information from pairwise. And I actually talked about this at my ag biotech talk just last fall. But what's really interesting about this is, and this is the interesting thrust of how some people think about CRISPR is that our store shelves and the food we eat, sometimes there's a trade-off of being able to produce a lot of it and have it truly cheap, but then it loses its nutritional value versus having nutritious food that then costs more and then people avoid. And so what they do is they look at the fact that brassica, again, this is the classification that includes musters and things like canola, that they're very healthy, but people don't eat them. Who here enjoys mustard? I mean, in terms of it being your leafy salad. And the biochemistry that behind this effect is well known. And so that's what I have here in the top part of the diagram is there are these compounds, these plants build up a compound that category compounds known as glucosomalates. And glucosomalates are these relatively innocuous sugary type compounds that are sitting in the plant, but in response to sharing or tearing or other things that are happening to the plant, it activates in times called morocinases. And what morocinases do is they convert the glucosomalates into compounds that are highly pungent. And again, this makes sense. This is a way to ward off being eaten, right? I mean, you think about stink bugs or moderate butterflies, they taste bad and that's why we don't eat them. Same is true for the musters. And so what they're basically big accomplishment was to use a genome editing tool to knock out every copy of that morocinase so that now you have the same plant and it's just not pungent. That's actually highly nutritious. And so that's, I think, a really interesting example of how to approach the consumer space of trying to find and identify things that are highly relevant to human health, to what consumers are interested in and matching it with something that was otherwise an unavailable source for it. Yeah, we also do not eat milkweed. That's true. Sorry, my phone just dropped. Okay, so next slide. We're getting to the delivery advances. And so this is one that, again, I won't say if I'm a technical achievement is a particularly exciting one, but it does highlight and it's relevant to I think what's gonna happen in this field. And so this is some work that, again, if you look at the title at the top, Optimizing Electroperation Condition for Crisp-Pass-Mediated Knockout and Zona-Intact Buffalo Zygotes. So I wanna highlight that what electroperation is meaning in this case, and you see this in the diagram, is that electroperation is a way to deliver DNA into cells as well as protein and RNA. You can deliver that by creating electric fields. So that's what the term electroperation means. And what they're showing here from the diagram is that, again, you have to be set up to be an animal breeder to have a lot of in vitro for artificial subnation capabilities. But in terms of the science, once you've isolated those cells from an artificial subnation or from an animal, you can actually take that single cell and put it through electroperation to adding your CRISPR. And what they demonstrated here is that those electroperated, they optimize the conditions that can then go on to become fully intact, or well, they didn't demonstrate that in this paper, but in theory could go on to be a well-edited and then complete new individual. And so the interesting aspect of this is that this electroperation technique is super low cost, super low infrastructure. And so it opens up the availability. This is a proof of concept demonstration that you don't need to be a very tech heavy, tech savvy lab and build up a bunch of infrastructure to be able to do this as long as you have all the other capabilities of the breeding, artificial subnation, all those things going on. So I think that's relatively exciting. Now, something kind of similar has been worked out in human embryos and that one of the big challenges for dealing with human genome editing, at least in the germline, is that they become mosaic. It's very hard to actually at that single cell stage make sure that every single resulting cell is exactly what you want it to be. And if things are not what you want it to be, then that's really just not a good therapy because then every cell in the body ends up inheriting some sort of issue. And so this paper, we're showing that if you instead of taking the strategy of taking the zygote and trying to do a microinjection, if what you do instead is at the same time you are doing the fertilization or say injecting the sperm into the egg artificially and also deliver the CRISPR components, then you get much more uniform cells resulting out of that, that have to change you want. And so that's what the diagram here is showing. On the top line, you're injecting it after it's already been fertilized. And so you get some brown cells which are not what you want, the blue cells in this case are what you want. And then on the bottom example we've been doing it before for those or you know, co-injection with the sperm that everything ends up being corrected at least more frequently. And that's what the diagram I'm showing on the bottom rights, the column graph is that the percentage of blue goes up a lot compared to it being uncorrected or mosaic when you do it this particular way. So again, this doesn't overcome all the other issues that one might have with off-target effects or whatever, but it is a step forward time of understanding the right way to do this in terms of germline editing. Then the other thing that was kind of interesting out of this paper was this, when I talked before about double-strand brakes, that there are kind of two main ways to repair them. There's something known as non-homologous end joining and that can be mutagenic. And there's also something called homologous recombination. And the homologous recombination is usually how you want to repair it to the right version. And so in this cellular context, HR seems to work more often than in, say, skin cells or adult cells. And so you can actually have a better therapeutic outcome for what you want to do. And so again, this is, again, nobody's proposing that we start doing human germline because there are still ethical issues with that, but in terms of evolving the technology towards something that we know works efficiently, then this is, I think, an important step forward this year. Now, again, most therapies and most types of deliveries we think about are things where you have an adult, you're trying to deliver it to their somatic cells or tissues via an injection or a pill. And this is another, and that's always been very difficult with the components you have with Cas9. It's a protein, it's got RNA, where you might be trying to express it from DNA. It ends up being very complicated. And so there's been a continuation of advances going on with that. And so this is one that was actually pretty interesting where there were, the group used prime editing. So again, this is an example of prime editing where you're trying to make a precise type of change using a modified version of the CRISPR-Cas complex. And they showed that you can deliver it with a reformulated, what are known as lipid-viral nanoparticles. Sorry, lentivirus-derived nanoparticles. And so lentivirus, again, it's a virus. You can actually deliver DNA in it. But in this case, they're actually using this combination of virus and lipids, again, think about soap, as a way to deliver this into the eye and actually get effective editing within the eye. So that was in the mouth, though. That was just a proof of concept, but a good example of where the technology is going. A technology that I've talked about a little bit before are CAR T cells. So these are chimeric antigen receptor T cells. And there are some approved therapies for these for certain cancers, including leukemia, other blood cell disease, other blood cell cancers. But not for something that involves a solid tumor. And so we're finding ways to make these, this type of amino cancer therapy more effective. But so far, editing has only helped us make the T cells work better against non-solid tumors. And so what this group reported is that on the left diagram, this is kind of the standard receptor that you put on a T cell, and the jack underneath is what basically drives the cell to death. What they did in this particular case is you see the zip two R, there's that orange band and that blue band, these naturally bring the jacks together. And this comes together in a way that produces cytokine signaling, but then also doesn't produce side effects. And the fact that it works this way means that if you can target the T cell to the solid tumor, you can get this continuous activation to target and treat and kill the tumor cell. And so again, they accomplish this with genome editing. We'll probably see lots of just, and I'm also using this to set up something else that's interesting in target genes. A tagline mentions in the text chat that lipid nanoparticles are also a delivery vehicle and mechanism for COVID-19 mRNA vaccines. So again, in the old days, vaccines were just pure protein. They were peptides you could inject into someone and your body recognized that and they were able to use it for immunology before it got degraded. But something like mRNA is very sensitive. Cells and blood will degrade it right away. And so packaging it within some sort of lipid coat, basically like a little jelly ball is one way of thinking about it, that protects the RNA until it can get into, and it will get absorbed by the cells to go inside the cells and be effective. So that's a big part. So yeah, this is technology, again, not unique to the CRISPR field. So one of the other challenging delivery spaces for humans as well is the brain. The blood-brain barrier, as well as just understanding how to get things specifically to... A lot of therapies we talk about tend to target the liver because the liver just sucks up anything that's coming out through the blood and filters it and reacts with it. And so in this case, this group was showing that a peptide known as tax-taxal is something that you incorporate into the lipid nanoparticle. And then that allows you... If that then gets specifically taken up by the brain. And so what I'm showing this diagram, I'm going to grab my pink arrow here really quick for this one, is that this is showing in a mouse model the amount of red you see in the square is a background amount of red from this transgenic mouse. It's set up, it's called a cherry indicator. The red means, oh, editing has been accomplished. And so in normal cases, if you're not activating it, you don't get it. But what they're showing in this... What this group showed over here on the right-hand side is that when you use these tax-taxal peptides to target to the hippocampus, you actually get a whole lot more red cells. And so it's a demonstration, again, in this way that you can get efficient brain targeting with this type of delivery mechanism. And so I think that's something where... That's an exciting area because that's a tissue where there are some, and we have talked before, about genome editing as a solution for things like Alzheimer's or other plaque-based diseases. Yeah, Max, I think these are still just blood injections. I would have to go back and double-check, but I don't believe they're not doing something like injecting them directly into the brain or anything. These end up circulating. I mean, the blood-brain barrier, you have to remember, it's not complete, right? We obviously are able to deliver metabolite, it's in fuel and energy and cholesterol and other things to the brain. The blood-brain barrier is not 100%. There are things that are... It's selective. It's a selective barrier. So again, exciting space to work in that I think can be relatively powerful. Now, this is something where... When I've been talking about lipid nanoparticle delivery, so far, the field on the work has all focused on being able to deliver the protein and the RNA, which is a great advance, but so far, this other way of doing genome editing, where you add a template, where you add a specific sequence that you want to replace the bad sequence, that has not been possible so far because the DNA is not readily held inside those particles. So this is an interesting one. And then also, when it comes to thinking about lymphocytes or blood stem cell diseases, these are almost all these things that someone takes out of the body, modifies it, and then puts it back in the body, which is, again, an ordeal. What we actually have from this group is the demonstration that you can actually create lipid nanoparticles and target them and get them into the human lymphocytes in the body so you don't have to take them out, put them back in. And so that's what they're demonstrating here with these what are called anthophilic peptides that are specifically targeted to lymphocytes. But the other thing that this group also worked on was, again, taking these viral-derived particles and then packaging up the DNA repair template within the viral particles. And then this way, you can actually co-deliver the repair template, the specific replacement sequence you want to go in, along with your CAS protein and RNA, and thus accomplish a very specific type of editing outcome. And so that's what's just been shown here on the right-hand side in the picture, is that the E5-TAT, N7-TAT, that's showing the IAEA5K and E-POR, those are showing variations of the peptides and the guides for targeting those cells. And so the fact that they're getting those knockouts to work the right way, the farther the bar is going to the right, the more effective it actually is. So I think, again, an exciting thing, again, we're coming back to the idea of you can go in, get a shot, and then that's your therapy for your lymphocytes. Okay, so getting outside of the human therapy, I think this is an interesting delivery advance. I will say it's the combination of two ways of thinking about how to deal with plants. And there's nothing really particularly technically novel about part of this, but the basic idea here, and what they're doing is, they're doing genome editing in cotton, using what's called virus-mediated CRISPR-Cas9, and that's what's shown here on the left, is that if you have a plant, and it's showing it its normal green color, and then you use a syringe, which actually people don't actually use in this case, they do something called leaf infiltration. But what they're exposing that leaf to is essentially pathogenically crippled, but active plant virus. And what this plant virus can do is actually, once you inject it in one part of the plant, and they're showing this with the orange, it travels throughout the entire plant. And so now, what they're not adding, they're not adding the CRISPR components, they're only adding the RNA that goes to targeting. Now, so that RNA by itself, in the absence of the Cas9 protein, doesn't do anything, but it now has traveled throughout the entirety of the plant. And so what they're doing in this case is on the right-hand side, they're showing that you take that same plant, or that same species of plant, but it's already been made to express the protein component. It's expressing the Cas9. And you trim off some leaves and do what's called grafting. Anybody here ever do any like orange tree or other grafting? I don't think they're screening during this period of time, Esther. I think they are tree lovingly, and these are happy viruses. And so what happens is, when you take the Cas9 graft, which you'll see in the diagram is initially green, then because the base of the plant has this RNA virus, that RNA virus then travels up, and all the RNA is coming in contact with the Cas9, and all the plants get, all the cells are getting edited, and then as the plant continues to grow, again, this is where it requires an active live graft, then basically all the rest of the plants, including pollen and the ova, are edited. And so basically, yeah, so no, that's exactly what's happening is that you can, the base plant that has the Cas9, you know, you've already transgenically made that in some way, and then the RNA, I guess I'm not clear on your question. The delivery is specifically delivered by scientists using this modified R-vector system. So now again, let me say from a plant perspective, this is not particularly novel or exciting in the plant cell, like bioc and the commercial plant cell worlds. It's going to be very useful for certain set of plants. But the area where I think it's really interesting is that this almost could be a consumer product or for certain plant types and certain smaller companies, this ends up being a very powerful system to get your genetic variation and then get your transgenic plants where you don't have to spend a bunch of time making your Cas9 plants or trying to do specific transgenic expression ones. All you have to do is sit up in a very low-level lab, the RNAs, and know your targets. And so this should be a relatively easy thing that at a lower scale allows great genome editing within the space. And almost even could be like a consumer-level product. I think that's the type of thing where you subscribe to a plant service and then you get RNAs. You have the Cas9 plant and then, or you have, basically you can get these and do whatever you want to mix and match in different ways, I think, from almost a consumer level. So I think this is kind of an exciting one when you combine these technologies. All right. So getting into the, I think, some of the interesting genes or interesting modified versions of animals this year, here's one where, again, not a particularly new mode of delivery. So this is, again, lipid nanoparticles. These aren't vital to our lives. These are just kind of your standard straightforward lipid particles that is used to treat hemophilia. So as you know, hemophilia is the failure to properly clot your blood. And that's because you're missing good expression of one of those clotting factors. So you might have heard of factor 7, factor 9, factor 8. And again, this is something that was prevalent in the English royalty. Well, the reason, the clotting works with this very specific pathway of factors that tell the next factor to do something. But there's also, at the same time, it's in conflict with what's known as an inhibitory pathway. And so what the researchers targeted in this case is a gene called antithrombin. And so antithrombin is an inhibitor of thrombin. And thrombin is an inhibitor of the clotting. And so if you're inhibiting an inhibitor, that basically enhances the clotting ability within these OIs, poor clotting factor individuals. And that's what's being shown up here on the top right-hand side, is that the CRISPR strategy, that bar with a perpendicular bar at the end, that's a science scientist used to say that's an inhibitor. And when you knock out, when you use that inhibitor on the thrombin, which is showing as an inhibitor, then now you get more fibrin. And fibrin are the basic particles you have for clotting. And so this, and then what the diagram at the bottom is just showing the strategy, the mixing. You basically have the lipid nanoparticles. And it's showing the very bottom right-hand side. The fact that the liver is very highly colored is showing that the lipid nanoparticle delivery went to the right place, because these clotting factors and anti-clotting factors are all made in the liver. Yeah, no, I mean, since that she also pointed out something that, you know, from a strategy point of view, you can always try and activate an activator to get more activity. And that's something that's valuable too. Well, and that's what it's going to say to her, is that this is, again, similar to as the example we had before was hyperchlamia or sickle cell anemia. The strategy here is to pick something where the therapy is extensive, highly involved, can be expensive, is lifelong. And so, and it's hard, you know, getting clotting factors and those injections into these patients is a lifelong thing. So this is a great target where if you can do, and that's what they're showing here, a one-shot therapy, then that's a big benefit in terms of the patients. Well, yeah, let me say that in the early days, Nero is also pointing out that in the early days of any new technology, because these are not being produced at scale, these are not being delivered to patients at scale, you know, the cost of these therapies is actually still expensive. And I'll actually mention that again at the end. So target genes and modified organisms continue. This is an interesting one because it's coming back to this idea of trying to create resistance to a pathogen within a population of livestock. So again, anyone here who's a midwesterner, also I think California gets hit pretty often, you're always hearing about chicken or turkey or other, you know, commercial bird flocks where they basically have to call the entire flock because it migrating birds passed on avian influenza. And so that is extremely expensive. And it is, as Susan G. had to say, a foul disease. It's also particularly pernicious because avian influenza is something that can jump the species barrier from, you know, from livestock to humans. And those are like, when you think about H5N1, there was that pandemic influenza virus, that's something that can cross the species barrier. So the more we decrease the livestock being affected by these, it also protects human health. And so the research in this case was showing that there's a category of host genes known as ANPs. And these are important for viral replication. And what they were demonstrating in these birds is that if you knock out several members of that gene family, they don't support viral production. And so they were showing that, again, what they are ultimately doing are creating offspring that are missing these genes. And then what's showing the bottom hand slide, on the left hand, so the black balls are showing viral load and infection in wild type eggs. And then what's shown in the triangles are the knockout eggs. And on the left hand side, you have them with the complete knockout. And on the right hand side, so basically on the left hand side, the properly knocked out genes are showing no viral load. So again, you knock these genes out, the virus cannot reproduce, cannot be, it's not just in the eggs, but also in the whole animal. And then when you basically do some confrontation work on it, then that comes back. And so this is a really nice demonstration within this model of you knock out a gene that's in the host that's not important for how the host survives, and you reduce the ability of the pathogen to reproduce. And again, this is a talk about CRISPR and not a commentary about the way we do or don't treat animals. That's something we can discuss some other time. Now here's one that's really cool. And this is something that gets into a really kind of interesting use case of CRISPR where, as you know, spider silk is incredibly strong and durable. And that it's something that's tougher than Kevlar. And people have talked about trying to find ways to produce that scale for that type of protection and durability of a material, of a cloth material. And so, but you can't scale spider silk production. It just doesn't work. But we do know that silkworm does work very nicely, right? That's where the whole, you know, China silk road industry comes from. And so the CRISPR, and again, this is one, this is one where the science is a little bit beyond me, but they modified genes within silkworm to make them in a way produce spider-like silk. Yeah, exactly, Max. It's basically silkworms can produce at their silkworm scale spider thread. And what they demonstrated within the paper is that it basically has these really highly durable, better than Kevlar toughness and durability properties. And so, this is a really nice example. And this is an interesting product class in the use case of CRISPR, because you're modifying an organism for something that it produces externally. And we have some examples of this with, you know, producing insulin and bacteria or producing other pharmaceuticals in plants or yeast is used to produce a wide variety of pharmaceuticals or other products. But this is, I think, one of these interesting examples of, you know, silk is kind of some product class of we're taking, it's like milk in a sense. Milk or silk would be the best examples of an externally secreted thing that animals make that we use extensively. Okay. This is an example in plants of the same strategy of trying to knock out a host gene that's really, really important for a pathogen. And that's what I'm showing here is this is rice looking at basically a fungus, a blast, where in normal corn leaves, you get these sores. In the middle, you see this RBL-1 mutant, the lower case italics means it's a mutant version of it. This infection doesn't take hold very well. You get a little bit of this scattered amount of lesions, but they're not something that impact the whole plant very much. And then the CRRs are saying, oh, they took the mutant version and corrected it. So just showing that that is the gene that's important and relevant. And the graphs on the right are showing lesion area as well as relative fungal biomass. And you'll see that the RBL-1 mutant basically does not seem to support the fungus. And so the key point here is that the virulence factor was identified through a random screen. But the problem was this mutation makes the cotton also itself very stunted and not usable to grow. And not a non-commercially viable mutation. And that's what I'm showing here in this next slide is that compared to the wild type, you look at that cup on the right, that is the RBL-1 mutant plant. So that's not useful for making cotton. But what this group did, and this is I think a really interesting use case where they know this gene provides protection from the virus. And then the question is, could they find a mutant version of it? Not the one that they randomly found, but use CRISPR to keep working on finding ways to make different versions where you have what's called a separation of function. That you basically have both traits within this. That the mutation allows for the plants to grow healthily. You're not impacting the health of the plant, but it still conveys resistance. And that's what this middle plant is showing, that the RBL-1 Delta-12 mutant specifically is able to allow the plant to grow to healthy levels produced cotton. And then what you see in the right hand diagram, again looking at the amount of disease infection on it, is that the wild type has high levels of infection, but this RBL Delta-12 mutant has very low levels of infection. So what I think this opens up is the opportunity that lots of people can go back through their back catalog and plant or maybe even chicken or cow molecular biology. And anything that was a combination of, oh, it's a great gene for resistance, but then the host was less hearty. We actually go back and try and use CRISPR to remake those, to see if we can find something that does something like this. So I think that was a particularly interesting one this year. Although I think any molecular biologists would say, well, we knew how to do that already, but this was a good demonstration example. Okay, so another one, again, talking about the plant relevant world, is that, you know, I've talked about this multiple times, that a lot of the most interesting things going on in the plant CRISPR world is trying to make them resistant to pathogens, help them resist climate change, in particular droughts, maybe make them more efficient at nitrogen usage or, you know, fertilizer independent. And this, but there's also another category of biotech that's really interesting, which is highlighted by cassava. And so cassava is a staple crop in Latin America for many cultures, but it actually produces cyanide. And so the amount of care you have to take in preparing it is extensive so that people don't get sick or die from actually consuming a staple crop. And so what this group is demonstrating is that they've identified a couple genes that are important for cyanide production, and that's what the graph is showing, the y-axis is how much cyanide is being made, the aqua graph is your wild type, and then they're showing two different mutants. And where you see on the bottom part of the graph where there's two Xs, that means they knocked out two genes important for cyanide production. And in all cases where they knock out both those genes, there's very low, there's undetectable amounts of cyanide in the plants. And so again, an example of a genome edited, targeted, we're basically making something more edible so it doesn't kill you. So it's kind of like, in a way, the conscious green mustard salad from Paralyze, but this is talking about something that's poisonous as compared to something that's just distasteful. So I think, again, also helping a stable crop. Yeah, do you not take a Cassava martini from someone who is not familiar with how to prepare it? Here's another interesting one. So I'm talking before about genome editing being important for modifying maybe plants or bacteria to help with climate change. But these kind of out there unique organisms are not good lab models. You don't necessarily have the tools to be able to work with them very easily and right away. And so what this group is demonstrating is that they took a methanotroph. So methanotrophs are things that can actually derive energy and incorporate carbon from methane. So again, you can convert methane into organic material. And it's better than having methane because it's a very powerful greenhouse gas. And so what's being demonstrated here, these are just various genes that they're knocking out in various isolates of the bacteria. So the fact that you see lower bands compared to the upper bands means that it's an effective genome editing technology. So I think that's exciting. And we'll see a lot. And being able to genome edit these things to be able to do this methane incorporation and processing to make them better. Because these are all good. These all can do this at a biological scale for them but not at a commercial industrial scale. One of the, like I mentioned, CRISPR so far has been thought of as a platform in an editing system derived from prokaryotes. There was some work in 2020 demonstrating that you can look and see these sequences in eukaryotic organisms, but no one had demonstrated that they can be programmable within a lab setting. And so that's what this group is showing is that this category of proteins that was fans ores were eukaryotic CRISPR looking proteins that in fact you can turn them into actual genome editors. And so that's what's showing up in the diagram is just kind of this idea that these have radiated out. You have some in eukaryotic organisms. You compare it to the structure of the known prokaryotic ones. They demonstrated that you can in vitro get it to cut DNA and then that graph H is the demonstration of targeted edits in human cells being made by a eukaryotic derived CRISPR protein. So whether this ends up being something that has new features or new availability or teaches us some new tricks for how these work, that is unclear but that possibility is out there and also kind of changes the intellectual property landscape as well. So we'll see, but it's a very interesting and exciting development for the CRISPR this year. Now the other thing that CRISPR has been useful a lot, again, I've mentioned mostly, and this is one of these different types of tools for cutting DNA, but because it has this programmable way of guiding and pairing with target nucleotides, it can also be used for detection. And so what I'm showing here in the diagram, again, how this diagram works is not important, but they're showing that with CAS 13, which can natively bind RNA, that if you add in some extra chemistry to the RNA and you expose it to a tissue sample or cellular samples, that basically you can detect whether it's present there or not. And quantify it. And so what's unique and new about this is that most times we are trying to detect the expression of something, how much RNA, not the DNA, but the RNA is present in the tissue sample or cell, you have to go through some extra processing steps. And so they're demonstrating they can do a direct detection of RNA with an RNA CRISPR protein to make that much more sensitive as well as quantitative. And then lastly, this is something, again, on the border I think becoming a consumer level product, but I still have in the new tools as compared to something on the shelf is, if you're allergic to nuts, that's really bad. And so the presence of nut material like peanuts or hazelnut in any sort of processed food, again, because it can, even though your input is not nuts, if it's being produced in a factor that also makes and processes nuts, small amounts of it can be rather lethal to people. And so what this group developed was using this type of targeting and detection of DNA, again, this is DNA, they're detecting hazelnut DNA in processed foods, and it's something that can be done outside the lab. And so this is something I can imagine will ultimately be something that maybe companies or regulatory agencies will have some version of this as a way to detect food, but maybe something that can also come direct to the consumer. Consumers have a version of this where it's like, oh, I'm allergic to nuts. Let's make sure that this food doesn't have any of it in there so that you don't have an allergic reaction. So there you go. Something I think will be a rather interesting development. It's something that really puts CRISPR as a technology in the hands of people for something that, you know, you probably don't know how it works. You just get a color, but it's derived from CRISPR. Okay, so cautionary tale. So as I mentioned, and I set this up early on, is that sometimes things go wrong with CRISPR. You're making breaks in DNA, and that can be repaired incorrectly. You might be making breaks in the wrong spot. And so this is something that the field is trying to take a look at how often this occurs and how dangerous it is. And so technology I've talked about before is that, you know, HIV is a retrovirus. And so it inserts its genome into T cells and then produces more virus. And so the strategy with CRISPR has been, if we can just keep mutating those integrated virus genes in the cell, we can eventually inactivate it. And that's been shown to be rather feasible within a therapeutic range. But the question has been, if you're making these breaks, can there be breaks that go outside what you're trying to do? And so, again, this is a complicated looking diagram, but the point being is, if you look at the diagram, the black represents the viral DNA and the green represents the host T cell DNA. And that sometimes when you're making these breaks, it's not just in the back, sometimes it infiltrates into the green. And so that's what this group was demonstrating, is that the strategy of breaking DNA within the virus can lead to things outside of the virus. From a quantitative point of view, it looks relatively small, but it's not something that I think would stop the technology if you're otherwise facing a lethal disease, but it's something to keep an eye out for. Then the last cautionary tale. Like I mentioned, and I don't have good diagrams for this, so from the paper, but we talk about base editing, being a way to do stuff with CRISPR that is not making double-strand breaks. And the theory behind that has always been, well, if you're only making nicks, you're not going to create breaks in other parts of the genome or as your target site. And so what this group was showing is that that's not entirely true. It's definitely a much lower level of double-strand breaks than a double-strand break cast protein, but these base editors still can have some degree of double-strand break and genotoxic effects. So again, something that people are still looking at. So again, probably still keeping it in many ways from being, or you have to basically make sure that your particular way of doing it, your particular guide RNA, the particular target you're using is still not creating these side effects. And then I think one of the exciting things that came out this year, a publication from The Galaxy that you can in fact, I'm just kidding. This is from Guardians of the Galaxy 3. This is the High Evolutionary with Rocket Raccoon. And what's specifically mentioned about this villain, what's specifically mentioned about Rocket Raccoon was that he was a genome-edited organism. And so I think, again, if you do a search on IMDB for movies that mention gene editing or things that mention CRISPR, it's actually still relatively few. But I think that this is, from this year, an example of a high profile movie that made hundreds of millions that actually kind of mentioned genome editing as a part of it. So anyway, I think it's something that we'll probably see more and more of it in movies or at least mentioned without going into much detail. So anyway, in summary, 2023 I think probably has been for the consumer and for what we have on the horizon for patient therapies and plant biotechnology probably the most exciting year. We have an approved clinical therapy and as I put parenthetically, a rating insurance coverage. The current estimated cost of that sickle-cell anemia therapy, cascading, is $2 million. So it's not cheap. CRISPR food is now in the U.S., something that's, again, we broke through that barrier to have the first one. And then we have a continued variety of specialized plants and animal applications. I think some of these things where we're inducing more pathogen resistance, but then also taste, I think it's really important. And again, a continued variety of tools outside the lab, things that are putting what CRISPR does in people's and consumer's hands that are not technical scientists, but is, again, something that's using it for some diagnostic, or in this case, a food source. So thank you for your attention. I am happy to stick around and answer any questions from the chat. I'll take a quick look. If there's anything you said in the chat that they didn't kind of address while talking, then please repost it or send me an IM.