 All right, welcome everyone to the first science circle talk of the decade of 2020. Hope you're all excited to listen and hear about some of the exciting stuff that actually happened last year. But of course, this is an ongoing area of development that is genome editing. So what we talk about today will really in many ways be about the future of the technology for us. My name is Steven Gager. Here I am in second life is Steven Zootfly. My job in real life is I'm a senior research associate at Corteva Agro Sciences, specifically working in the basically the technology development area specific to genome editing as well as plant transformation. So that's my background and what I find also interesting and what I really liked about preparing a talk about CRISPR in 2019 in general is stepping a little bit outside of my narrow area of research to tell you about the things that are exciting. Now, some of this might be biased towards the things that I think are most important, but I've tried to make it what I think really are the highlights of 2019 in genome, genome engineering. So with that said, I'll get started with my talk after one brief mention that, of course, a disclaimer. I am a researcher at a company that is involved in this area of research trying to commercialize or related to commercialization of products. So just understand that potential bias. And then I'm not here representing the company's positions. I'm not here as some sort of delegates or official capacity for the company. The opinions I express will be my own. Then additionally, nothing that I talk about today should be construed as an investment advice or forward looking statements for the company or even kind of maybe some of the other research that I'm talking about that does have partial interest. Again, if you go investing, putting stocks in these companies, don't blame me. There are other things going on. So the basic structure of my talk is I'll give a reminder about the background of how genome editing works. And then I'm going to talk about, I think, some of the most interesting, likely impactful technology advances. Talk about some of the new tools that have occurred. So people are very focused on something known as Cas9. You'll always see Cas9, but there's actually other stuff going on in bacteria that we can take advantage of for genome engineering. So we're going to touch upon a little bit of some new developments in intellectual property and ethics. And then finally, the bulk of the talk will be talking about, I think, the most interesting development we have right now, which is the initiation of clinical trials for therapies using CRISPR-Cas9. So that'll be pretty exciting. I'm kind of moving away from talking about some of the model genes and model organism research that's happened. There's been a lot of great stuff going on out there in terms of other genes targeted, stuff going on in mice or other organisms. And maybe we'll come back to some of those in a future. Anyway, with that being said, I also want to just make a point about the way I've structured this talk, which is in order to be faithful to the research that was published or maybe press releases, I've included a lot of text from the original publications or press releases. Do not feel obligated to read those on the slides. And I will be highlighting the things that are probably most important. There's a PDF that will be on the website that you can always go through those more carefully. And then the other thing to keep an eye out for is if you see some green text, those are kind of my upshot take-home lessons for any given slide. So if you want to focus on those two, that is just fine. All right. So as a reminder, if you look at the slide, this is a review from Geofford Doudna, one of the developers of the important technology of CRISPR-Cas9. And what I want to highlight here is just the basic idea that on the left-hand side, you see a gray outline of the protein Cas9. And what it's doing is clamping around some target DNA. That target DNA is in black. And what's also in this complex is a blue piece of RNA. And this is what binds the RNA to the Cas9. And then associate with that is this red RNA known as the spacer. And what the spacer plays an important role in doing is guiding this gray protein to that black DNA. So what you'll notice is that the black DNA, the black strands are split into two. And then the red is hybridizing. In other words, the bases of the RNA are pairing with the bases of that target DNA. And that's what allows us to specifically locate to a specific sequence or location in a genome. And then you can do stuff. So classically, the Cas9 in and of itself contains an endonuclease activity. And that's why it's showing little scissors on the diagram. And that you can cut DNA and then things can happen. We'll talk about that. Now, one of the other developments that has, it was discovered a few years ago, is that Cas9 is not the only protein that does this that are derived from bacteria. There's something known as Cas12A, which you may have heard called CPF1. Same basic idea that you have RNA hybridizing to DNA. And then that's cutting some location in genome. And then on the right is another category of these enzymes that actually doesn't target DNA. It actually targets RNA. And so the ability to do this type of editing, you don't necessarily have to target the genome. You can actually target the messengers of the information. And in some applications that might be useful or also allay fears about genome, you know, germline editing. So what are the things that scientists and researchers have decided to do with this? And, oh, sorry, let me make one other statement that the reason bacteria have these, it's just an immune system. And what's really unique about this type of immunity for bacteria is that they can develop a memory for things they've seen before. And that's a very powerful thing, like our, if you've had a vaccination, you typically don't get infected by that thing again. And so the same idea happens with bacteria. So these are exquisite. And the best way to defend yourself from viruses is by just cutting their DNA or RNA. Now, again, continuing from the same review, Dalna describes how the things that we can do to target that target some sort of effect in a target organism or cell is that, again, cutting is something that makes sense. And this other category of things developed by David Lou and some others at Harvard, where instead of cutting DNA, they make small mutations that then get repaired in a way that's different than what you had before. So it's called base editing. You can also locate genomes by lighting them up with GFP, green fluorescent protein. You can also turn genes on and off if you take out Cas9's cutting activity and then put other things on it like transcriptional activators or transcriptional repressors. You can turn genes on or off. And again, you can do some of the similar types of things with RNA as a target. Now, Vic asks a little bit of a side question. I'll address it, which is when, which he asks, how do they store that memory information in their DNA? And that is correct. What they do is when they find a foreign invader, not only do they chop it up in order to inactivate it. They also take a couple pieces of it and basically take that sequence and re-encode it back in their own chromosome as a part of the CRISPR locus in order to then re-recognize it and have that always available. Again, I don't want to go through too much of the bacterial part of this because we have so much exciting stuff to talk about with clinical trials. So the, now this is a review from Platt, who is describing, reminding us how we think about genome editing with Cas9. And there are two ways that we've been doing this in general, which is one, to let Cas9 make a double-strand break and then either let the host organism repair it in a way that you create mutations. Which, again, it's very hard to direct the types of mutations that occur. And sometimes these are just small deletions. Sometimes they can be small insertions of just a few nucleotides, a couple bases, a couple A's, G's, but we can also provide a template. And that's what you see in this red DNA is the ability to say, well, we want some other different sequence in this location and then insert that. The problem with that is that in general, it's very inefficient. Now there have been some developments that might find a way to make that happen a little bit more efficiently in certain types of cells. But for right now, doing this insertion or HDR based repair is not something that we see in particular in the clinical trials right now. And as I mentioned earlier, this idea of base editing. And what the idea here is you basically take Cas9 and it says something that can make a double-strand break and it's something that can just make a nick. It only cuts one of the two strands of the target DNA. And then you also add what's called a deaminase. And deaminases are something that say, well, if you have an adenine, again the base pair A, you basically cut it out. Sorry, an amine. You cut it out and then other repair processes come in and switch it to a different base. And this is again taking advantage of the repair processes that happen in the host organism. So you notice here they're showing you can take a G on the top strand and convert it to an A. Now that again is also not particularly precise. It's not particularly always the same sequence, but it does work relatively efficiently. And the other important thing about base editing is it avoids making double-strand breaks, which can have, if misrepared, very bad consequences for the cell. And so again in these types of strategies where you're taking cells out of a patient trying to do genome editing and then put them back in, then this is maybe in certain ways less precise what you want to do, but also less dangerous. Now this is the new development. And this was the review covers this paper from Platt et al. Where they basically, oh sorry, from Anzalone et al. That, but this diagram describes a little more nicely, where instead of just trying to cut or nick DNA and let the guide is to include on that RNA that's attaching to the Cas9 is to also include a reverse transcriptase. And what reverse transcriptase does is it takes an RNA sequence and converts it back into a DNA sequence. And this is something where if you know about, you know, human immunodeficiency virus that goes around infecting cells as RNA, but it encodes reverse transcriptase so it can turn into DNA and then incorporate into and then become like a permanent part of target cells. And so using the same idea is that if you include this red template sequence as a part of your RNA, reverse transcriptase can come in. And then because of the way the Cas9 is making nicks and because of the way cells prefer to repair these types of extra sequences, you can actually code for a specific repair process, a particular outcome that you see in red. And so, again, in summary, what we saw in the previous techniques is you are likely to get outcomes from the activity of Cas9 that you can't control. And the main idea here is that now you can actually frequently control the outcome of the edit. So again, Vic asks a good question about, you know, what environment does this all take place in a lab of safety protocols? Yes, you know, anything that any lab that's ordering supplies or getting money from granting agencies go through and have safety protocols. There are institutional review boards and institutional, you know, health and safety committees that cover all this type of thing. And so this is so this technique is called prime editing. So the idea that you can prime off the RNA and determine the outcome. So here's just one quick slide from the original paper. I don't want to overwhelm you and you don't need to read this in too much detail of close. But what I want to demonstrate from this paper, and this is what I have written in green, is that if you have any base close to your target sequence, which is a C and A, a G or T, you can turn that with pretty high frequency into any of the other three bases that you want. And that's kind of what that top graph is showing is that all those blue bars are demonstrating a pretty high efficiency where you're replacing one base with another. And then on the bottom graph here, the slide F, what it's showing is actually doing more than just trying to change a base from one to another. It's actually inserting or deleting certain amounts of small sequences. And again, in certain types of editing situations where you maybe want to change a protein or some target gene, those types of changes are more important than a single substitute. So any any quick, this is a good place to pause because I think prime editing is a great time to say we found a way to more precisely make the changes we want. So any questions about this so far. All right, so everyone's feeling pretty good that they're understanding the base, the base concept of what's exciting about prime editing. All right, great, then we will move on. So throughout all of the development of CRISPR-Cas9 technologies, the idea that you can hybridize and target a particular sequence. One big question is something that has been observed is you can accidentally target and have outcomes at sequences that are similar to the one you're targeting, but are not the one you're targeting. And so, for example, if you have one or two base substitutions, Gs or As, you can actually end up targeting and breaking or editing some other gene or some other location, the genome. And that could be again, right back there. So this is known as off-target targeting. And so the data coming from this paper, this is something from, again, Young et al, which is actually my group at Corteva AgroScience. And the upshot here is showing that when you include Cas9 and target corn embryos and then look at actually sequence verify all of the other related sequences that what you'll see in that green box is you get, again, in this particular situation with Cas9 being delivered as a protein, you get zero off-target effects. And I think that that's an important thing going forward when we talk about the safety or efficacy of Cas9 and genome editing is that we can be relatively sure that with certain conditions they do not target other stuff. So, again, there's a lot of conversation in the local chat about the Chinese scientists who did genome editing. I will talk about that later. There was actually a December 30th outcome. So there's very recent data. I didn't even have it in my talk until I started preparing this just last week. So we'll talk about that as well. So, but was also interesting from this group and looking at corn embryos and targeting in the next slide here with table three is that if you do a very good job of picking a target site that has very few other off-target likely sites. Again, there's a likelihood that an off-target site will be hit based on how similar it is to the original. That even with, you know, basic vector-based expression of Cas9 and the guide that you still have zero off-targeting effect. And so I think this is a very important thing when you think about genome editing that, you know, it's actually much a little bit safer than we thought as long as you're rationally designing your strategy. There's some similar techniques that was also published this year looking at base editing off-target effects. And so also in 2018, again, a little bit earlier at the end of 2018, so I actually kind of missed it when I was hitting the holidays. There was a very nice paper done in mice showing that off-targeting effects was relatively minimal in mice. So I think these are, again, kind of the new developments that we have that are, I think, the new technologies that we have that are actually pretty cool in the CRISPR-Cas9 area. All right, so moving on, CRISPR-Cas9 is not the only way that bacteria try and defend themselves. It's actually a relatively small percentage of these CRISPR systems. And so if you take a look at the top left in the diagram in Figure 4, what you see there is this complex of blue, pink, green proteins along with an RNA. And what that actually represents is a protein complex known as Cascade. And that is a CRISPR system known as Type 1, where there are multiple proteins that do the functions of going and finding or, you know, finding the RNA, going to the target sequence, and then they separately recruit yet another protein in order to do the cutting in the natural bacterial systems. So what the Picker-Oliver group showed is that if you take this complex and add, as Doudna mentioned earlier, transcriptional activators, then you can turn on the expression of endogenous gene. And so that's what's shown in the graph below it is the basic outcome data where if you're measuring the amount of expression of the IL1-RN gene, you can actually get increased response expression when you're specifically targeting its promoter. And so that is an exciting development that we have other tools and techniques that allow us to do these types of homing in human cells. And then what's shown here on the bottom half and the bottom right-hand side, those little odd, orange-shaped oblong things, those are corn embryos that you can dig out of a kernel. And then you can use gold particles accelerated at high velocity to deliver DNA. And so what you see on the right-hand side is using Cas9 with a gene activator to something that turns on a pigmentation gene. And what you see in the middle is that is also this cascade complex that, again, attached to various gene activators that also accomplish the same goal. And again, the left-hand embryo, that's just a control showing that without these types of gene delivery systems, you do not have any activation of the pigmentation. So again, an exciting tool that we can use both in mammalian systems as well as corn. I'll pause because Vic asked the question, are we learning a lot more about turning on or off genes with CRISPR technology? And, you know, this technology, because if you want to turn a gene on or off permanently, you would also have to include that whole trans gene and embed it and keep it turned on. So it's not a hot area of research for, say, gene therapy because that becomes a transgenic. It's also, again, for plant-based agricultural products, also not a thing that people are focusing on. But it is a very powerful way to change stuff. And the one thing that some people are working on are things that change the epigenetics of a target gene. So maybe you can get some degree of permanence where you transiently change the promoter status and then you can turn it on or off. And so those types of ideas are being explored. But I wouldn't say that there's anything that, you know, is close to product development right now. But you can use that to learn very interesting things about these systems, about target organisms. So, again, the work with the embryos, that's published by me in my group just this year. So, again, that's strange genes on or off, but one of the other applications we're really interested in is the ability to, of course, make genetic changes to target chromosomes. And so two other groups have also been working with these cascade complexes to accomplish the same thing. So the Dolan et al group, they took this cascade complex and took its normal endonuclease, you know, as Cas3, and put this into target human cells. Now, the reason why Cas3 has not been really a focus of genome editing is because it just goes around being like a Pac-Ban chewing along DNA. And that's what the diagram is trying to show here too, is that Cas3, it jumps on, once it jumps on DNA is activated, it just chews it up. And so the ability to do precise genome editing is limited when you're letting Cas3 perform its normal activities that way. However, there are applications where this may be useful. And so, again, they published that this works. And it's important to think that, hey, maybe if you can deliver and get this to work, there are other ways of, again, modifying target DNA, maybe base editors. Now, the Cameron et al paper, and this is interesting, this is an older technology that's been around for a long time where they can use this dimerizing endonuclease, known as Foc1, which I'll just spell in local chat is FOK1, where if you can provide these coming together on top of DNA, then they'll make cuts. And so what they showed was that with a cascade complex attached to Foc1, and then targeting some ticker sequence, you'll see that the bars that are high up there, that's showing the editing efficiency of this type of complex. So again, this is an example where, again, maybe Cas3 is not going to help you do what you want to do, but being able to deliver a different type of endonuclease will help it work. And what's really interesting about this type of technology is that because you're guiding cascade to different parts of the DNA, the sequence specificity is, you know, log fold higher than what you have with just a single guiding Cas9. And so that idea may be useful. And then just one other thing that, you know, we may or may not see out of these systems is that because there are large protein, there are lots of different ones involved, maybe you can attach different types of moieties to do multiple things at the same time. So combinations of hypermethylation, transcription activation, base editing all at the same time. And so they're, even though they're bigger and maybe less, maybe a little more unwieldy in terms of transgenics or other delivery systems, they may have other uses that you can't do with Cas. So the other new technology that has come out is the discovery by two different groups with two different independent systems that transposons use CRISPR systems in order to jump around. So let me just mention that transposons are segments of DNA. They're also known as selfish genes or sorry, selfish DNA or junk DNA or mobile elements are different names for them, where they live in a genome, but they're more like parasites that are jumping around. And what the copy group at all demonstrated is that there's a naturally existing cascade complex that associates with within the transposon and then uses its transposase, the enzyme that accomplishes transposition to actually insert the transposon in other genomic locations. And so this idea means that maybe we can do payload delivery that's targeted. Again, we can exact the system from liquor biology to in a more safe way deliver payloads in particular genome locations. And so that would help avoid the dangers of making your canonical double strand breaks. And then there's another group that showed these single proteins again the CPFs to cast 12s accomplish the similar type of transposition. So again a little more of a simplified system. Now let me say the caveat to these this is very exciting. But the caveat is that this has not been demonstrated to work in eukaryotic cells yet this so far right now is limited to prokaryotes. But again, something that is potentially very exciting useful as we move forward. And then the final new tool that was something that came out from, you know, doubt and associated researchers is they went database searching for more cast proteins. And one of the ones they found was this cast 14 that Vick so you asked about junk DNA remind me at the end because that's a little bit off topic. And I can unfortunately go on too long about it that these cast 14 proteins. They also perform CRISPR so activities and what they able to show was that they could get single strength DNA cleavage with a guided RNA. That's very efficient. That's again what you're seeing here in terms of this diagram is the red bar or so the red dots representing single stream DNA cleavage. And so the y axis is how much of that is being cut up. And then the x axis is over time. And they're showing that multiple ways with the graphs, and then actually showing the gels are showing that you have DNA that is a high molecular weight. And then with the activity this protein, it turns it into a low molecular weight so things that are lower or at the bottom of the gel or things that have run faster on the gel and are smaller. Again demonstrating that this is yet again another protein that can accomplish these types of tasks. And then again also the Vilnius University group, Virginia's cisness also basically found that when you take the same protein and find some slightly different conditions in which to incubate it, that it actually does also work as a double strand DNA cleavage protein. So again very similar to cast 9 in the sense that targets DNA, you can guide it to a particular spot and then makes a double strand break. Now what's most exciting about these really small double strand break effectors is that because they're so small, they can fit more easily in adenoviral vectors. And adenoviral vectors are really the most feasible way that right now we do DNA delivery in human cells. And so the cast 9 by itself is a pretty big protein and takes a lot of space on a vector in order to express it. And so the ability to do this with a smaller protein that cast 14 represents may allow more clinical applications. So George let me get back to your question a little bit later as well because that gets into a different question about evolution and mutations. So that's kind of I think some of the interesting technology developments and the new tools that we have in this field. I'm going to move on to the next half of the talk where the thing that almost always comes up when people are thinking about Christopher cast 9 from a commercial, from especially from a commercial point of view and how are these going to become clinical trials or how these become therapies or how are we going to use these for other types of things is the intellectual property space. And so right now, the Broad Institute, Fang Zhang as the inventor are the ones that hold patents and the ability to license this for use in commercial applications in eukaryotes. And there and I've talked about this in the past there's been a history of UC Berkeley, the Sharpentier and Doudna groups that, you know, there's been an ongoing saga between the two of who actually owns it and from whom does any commercial entity need to get a license. And so there was an interference last year where Broad won, and there was no declared interference. However, the US Patent Office actually turned around this year and without being prompted by any particular lawsuit or any particular court case decided to reopen the interference. As related to a specific aspect of the technology. So one thing I've mentioned is that this RNA you use RNA to guide the protein where you want to go. And there are different ways to engineer that it's naturally a two piece system. And how Doudna demonstrated, and I think published first in eukaryotes, or sorry in prokaryotes and bacteria, was that if you can conjoin these together with an intermediate linker sequence, then it works better. And that that is, you know, feasible to do. I think the interference has been reopened that in terms of using that aspect of the technology of doing this joined guide sequence, the single guide sequence that feng shen may not have the novelty for that one. So, once we start talking legalese, I start getting lost. But let's just say for right now, in terms of the way most people use this technology, it's entirely possible that in the upcoming years, there will be a new ruling that says the Doudna group actually is the umbrella patent for the use in eukaryotes as well. So we'll see we'll see how that goes. And of course, other news was also kind of mentioned a little bit earlier in the chat is that this Chinese researcher who performed genome editing in the embryos of two humans, and then implanted them that led to live births. All right, so what we're talking about here is actual attempted germline editing of a couple's offspring. Well, actually several couples, several couples offspring. In order to basically the attempt was to make them immune from HIV infection. And I've talked about you can go back and look at my old talk, my old presentations were talking about the details to the gene and what they tried to do, and maybe other larger thoughts on the ethics. But this was a development that just happened December 30th, that he was found guilty in a district court in China of being guilty of different crimes. And the crimes focus around the idea that he conducted this research poorly in terms of ethics, informed consent and institutional review. And then maybe the larger broader category of saying that gene germline editing is not considered ethical by the science community. And that, again, the terminology to quote here is that rashly applied genome editing. And I think it's very fair to say that if you look at the outcomes of his genome editing, that both of the individuals that were born ended up being heterozygous for the intended mutation. So as a treatment, it was not effective 100%. In fact, it's only 50% effective in terms of targets. And then in another sense, essentially 0% effective in terms of it being a useful way of being able to resist HIV infection. So I think that that is would go into it as well. And I think fairly enough, a lot of researchers in the field have criticized him for not per se being ready and being 100% effective. So anyway, I think that's a new development. You know, there's been a lot of talk this year, and I've seen various headlines, but I didn't get into it for this talk of different scientific communities trying to really come up with what is the ethics of germline editing. And I think that that's going to be a big question. Maybe the 2020 year review will have a better conversation about what people are doing and the decisions. I did see in the local text, I think also there's mentioned a Russian researcher who went online to say, I'm going to do germline editing. But he actually, I think a couple months later said, look, the germline editing I'm talking about doing is with scientific review, ethics review, the permission of my government. So that made some headlines, but I think the follow up to that was less sensational than expected. All right. So let's talk about the interesting gene targets. And so one of the things that made the rounds, and this is probably something you may or may not have heard about. But definitely was making the rounds in the genome editing community was basically an oopsie. And so let me talk about what was attempted what they were attempting to edit, which is cattle. And that is that normally when you have a large herd of livestock of cows and bulls, the fact that they have horns means that they can damage each other they can damage the handlers. And so one thing that they typically do is remove them surgically at a young age. Now that's apparently not a comfortable process. It's considered inhumane by some people. And so the idea of genetically modifying them to not have horns in the first place would be a more humane way of doing this. And so there's a naturally occurring gene variant called pulled that is they don't they just are not born with horns. And so there were attempts to gene edit this and this was actually published back in 2016, they were planning on trying to turn this into a breeding colony. So that, you know, then the genome, the gene, the gene edit could be disseminated. So the first company that did this was recombinetics. And then the project was actually taken over a UC Davis. And so I have some web links where you can get familiar with this background and everything. Now, what came out this year that was published in two different sources was somebody analyzing the the publicly made available sequence of these individual. I think there were bulls. So, and what they showed here. And let me just say, let me walk you through this diagram for a second, which is that anytime you're trying to deliver a template. This is what I mentioned earlier on is that, you know, to actually put sequence in that you want, you have to add DNA to the whole to the whole whole mix. And so on the left hand side, what you see is a vector and all the stuff that has an orange shade to it that is backbone to the vector and not that's bacterial sequence. And then you have the targeted integration trying to do that's the other colors. In the middle, this is what a normal looking chromosome looks like that's not mutated. It's basically called the horned allele of the gene. And then what this group was finding was that on the right hand side, the journey genome edited bulls were heterozygous. Now they both had the pulled version of mutation so that on the left chromosome you see up there you see the gray, the green, the blue and that's what you had from the delivery vector in one chromosome. And what that did was to basically recapitulate the natural mutation that led to this this phenotype. But what you see in that little right chromosome is a whole bunch of orange sequence and then another copy of the green and blue. And so this is a very aberrant unexpected repair outcome. But the significant difference in terms of that left chromosome being the intended edit versus the right chromosome is it becomes a transgenic animal. So the fact that there's foreign inserted DNA means that the regulatory process that these animals go through is vastly different than a regular genome edit and at least most countries and particularly in the US. And so that's one thing that people are very excited about what genome editing is that you can move away from a regulatory schematic where you're in that you have to analyze inserted DNA from other organisms. And so that's this is something that basically kind of power through the community. Now I will say the UC Davis group also published in Nature Biotechnology. They were looking at the inheritance pattern of these chromosomes from a mating and they were looking in the offspring and they did publish that there is this extra DNA, which was missed in the original 2016 publication. So this is not something that as far as I can tell they were trying to hide. There were just some kind of minor experimental mistakes that were made. And these are difficult by the original original group. So the thing that's important to recognize here is that when these types of mistakes can lead to additional scrutiny, but it's also very important to make sure that you do everything you can to confirm a very carefully detailed types of edits and changes that you're making. And so we might see some sort of additional regulatory scheme, even when what you're trying to do is not transgenic. It's your kind of accelerated breeding editing schematic, but you need to make sure it's not other stuff hasn't happened. So that was kind of an important development this year. But I'm sorry, let me go back to say one thing that there are several examples of livestock, both in terms of cattle that are more heat resistant. There's also trying to keep pigs from hitting puberty in order to remove pig taint from pork. But these are, this is a very active area of interest of trying to genetically modify livestock in order to, again, become a better product or to make the handling and the growth and the human, you know, how humane we treat them better. Okay, so let's talk about the final part. Which are there are several clinical trials that have been published and talked about in the United States and Europe. There are a quick caveat that I do know of clinical trials that have been initiated in China for a variety of different diseases that involve CRISPR-Cas9, but they're kind of hard to track down and to really be able to say much about exactly what they're doing. So just to say that, let me just say that this is an interesting year for clinical trials, but we'll be focusing on what's happening in Europe. Okay, so the link that I have on here, which again the PDF, this will all be made available on the website to make it easier to get to these links. It gives a nice overview of these different clinical trials for this year. But I'll go through them in more detail. So the first one is sickle cell anemia and beta thalassemia. So, you know, for, for us to be large organism, large organisms, we need to be able to transport oxygen very efficiently and to pick up carbon dioxide very efficiently and basically dispose of that. And so hemoglobin is this incredibly fine tuned molecule that accomplishes this very exquisitely and very well. And if you have a sickle cell mutation, what that is is a single amino acid that changes how the protein folds, which also then changes how the whole red blood cell folds and can lead them getting stuck in small capillaries, joint pain, and then inefficiency at delivering oxygen. People with this disease without medical intervention typically died young age. There's also beta thalassemia, which is mutations in the beta chain of hemoglobin that also lead to similar types of inefficiencies in delivery of oxygen. Again, I'm not familiar with the all the clinical presentations of beta thalassemia. Now these are both relatively common diseases because they provide some degree of resistance to malaria. And so these are things that have naturally developed in, in areas of the world like in Africa and the Mediterranean, where malaria is spread as relatively common from mosquitoes. Now, anybody who has had a child or has born a child in their body has realized that there's a lot of extra things you need to do to deliver food as well as oxygen to you to develop. And one thing that has occurred in development is that the ability for the fetus to capture oxygen from the host mother means they have a different variation of hemoglobin, known as gamma hemoglobin or fetal hemoglobin. That's even that's good at grabbing the oxygen from normal adult hemoglobin. And so that's very important for developing embryo to get enough oxygen. Now what's been shown is that if you over express gamma hemoglobin in people who have beta thalassemia, you actually have some degree of resistance to the disease to or the symptoms of the beta thalassemia. And there are actually some natural variants of the disease that have occurred. So one thing I want to point out is that the clinical trials right now they're trying to alleviate sickle cell anemia or beta thalassemia are not trying to correct the gene that's missing. They're trying to over express a complimentary thing that is expressed in developing. They're not actually attacking the gene itself. So here's the basic press release from Vertex web page. And basically, again, we're talking about phase one clinical trials. So phase one means they're really just making sure that the treatment is not toxic and that nobody's that the treatment is not worse. Sorry that the treatment is not worse than the disease you're trying to treat. And so but at the same time that you're doing these phase one, you can also get some degree of clinical indications on a few patients that are being treated that are being treated. And so what they were showing is that consistent with what's been seen for people with natural mutations that lead to gamma hemoglobin being expressed. They're actually seeing alleviation of the symptoms in a couple of patients already. And let me go through the go through a little bit of the science background of how this particular genome edit is working, which is in this diagram, the small blue boxes on the left are the genes for gamma hemoglobin. And the orange is the beta chain. And these different blue boxes underneath this are naturally occurring deletions in the genome. In again, what's called, sorry, let me go back to this whole term. It's called adult persistence of fetal hemoglobin. I think it's the disease, something along that, that name. Oh, yeah, persistence of fetal hemoglobin. That's what persistence. So when this segment of DNA gets deleted, what's actually occurring and you see this in the very bottom diagram is that the red enhancers are things that help that that make genes get expressed at a high level actually get closer to the gamma hemoglobin. And so the gamma hemoglobin is getting expressed at a high level, even in the absence of the beta hemoglobin. And so the basic idea here is that you can do a gene therapy and target cells where you purposely delete intervening sequences in order to get the gamma hemoglobin to get over expressed. Now there's other companies that have strategies where they're trying to turn off the repression of gamma hemoglobin that occurs naturally. But that I don't think has hit actually hit the clinical trials as of yet. So again, an interesting strategy is trying to delete DNA in order to help express a gene as a therapy. Now this other one, this is coming out of your University of Pennsylvania that has a long history of trying to use immunotherapy to treat cancer. And again, here's your long text. Here's your long press release where I didn't want to have this as a part of the talk, you don't have to read it all. But what they're also doing is with this therapy they already have they're adding CRISPR to make it more effective. All right. So again, this is one idea where the therapy, the gene therapy that's already been developed already has gone through a regulatory process where they know it works. Just they want to enhance this effectiveness. And this is known as CAR T. So T cells that are chimeric, they've had they've had other genes added onto them or other. And what they're doing is they're encoding the T cells to recognize the cancer. Again, there's some sort of protein on the surface of the cancers. That's important. And then you're saying, hey, T cells, this is your enemy. Go attack them, destroy them and thus destroy the target cancer. And so this is the basic idea of the therapy. Now the reason it hasn't been as effective as one would hope is that there are other things that regulate T selectivity. And so one of these things is a protein on the surface of T cells known as PD-1. And what happens is cancer cells can typically have a receptor on their cell surface that interacts with PD-1. It then tells the T cell to not engage in immune action. And so this is a diagram from, again, RUP at all, which is part of the people working in CAR T's, where showing this in a mouse model is enotransplant type thing. This is where you put human cancer cells in a mouse and then trying to therapy on that to see as a model system. That you'll notice in the blue bars, or sorry, the ability to, what's known as the tumor burden, which is another way of describing the size of the tumor, that the pink bars where you have the tumor cell able to shut down the T cell, that burden increases. But if you have, but if you've deleted the ability to tumor cell to interact with T cells this way, then you notice the black bars, the tumor burden is low. And so the idea here is that for the CAR T cells that the UPenn clinical trials are delivering, they're deleting out the PD-1 from those patients. And this is an important thing. The reason why this is an immunotherapy, let's just go back to this diagram. You're taking an individual's cells out of their body, messing with them and then putting them back in. And this is a way to make sure you avoid lots of immune responses that the patient might have. Okay, and then the final clinical trial, this is something that is Lieber congenital amaurosis, which is basically a neuropathology in retinas, where basically it leads to blindness. Now, there's a whole host of genes that are related to this, but the most predominant one is something that is LCA-10. And so Allergan, again, if you have contact solutions or looked at that in the grocery store, Allergan is a name you might recognize. Then Editas Medicine, in collaboration with people who are the early developers of CAST technology. They're targeting this because what's interesting about it, it's really the first clinical trial of trying to deliver CRISPR-Cas9 materials directly to the tissue and then trying to edit it and have a clinical outcome. And so this diagram here explains the strategy. And so the reason that people have a mutate, the types of mutations that people have in this LCA-10 gene is they have some sequences between exons that cause aberrant splicing. So I don't want to go through too much detail of how, you know, central dogma of DNA works. But when you're making an RNA, you have to, you're making a bigger RNA than you need, then you specifically chunk it off in a way to give you a protein coding sequence. And if that process goes wrong, where you have either additional or missing splicing, you know, the way you're joining the components together, you don't get the right protein. And so the strategy here is to deliver CAST9 and try and edit these mutations that lead to the splice site. And so there are actually lots of ways you can try and delete that sequence, you can just try and modify that sequence. You can do a template-based repair, different ways to try and modify this splice site and get rid of it. And then that way you get the normal gene and protein being expressed. And so that's what their strategy is, and it's also in clinical trials, and they've shown that it is so far efficacious. All right. And then the final, again, this is a non-human application of genome editing technology. Again, this is not a CRISPR-Cas9, this is a talon, but it's related, where the big goal here now, or the big point, is that there are now people eating and ingesting from a commercially available source, again, gone through proper commercial channels, is high oleic soybean oil used in cooking. And so there's this group, Calixt is a Minnesota-based company that was founded by the person who developed a talon editing process. Again, my name is Dan Boytos. And they've used this to edit a gene, which we'll talk about in a second, where high oleic soybean oil basically has all these great advantages where you can use it to fry multiple times. It has a longer shelf life. You cause less residue buildup on the cooking utensils. And so they are really the first ones to have made a product and put it into the food chain, where, again, there are some restaurants that are using this in their cooking oil. And so it really marks, I think, a unique landmark of edited products getting into the food chain. Now, one reason why this has not been huge and big news, I think, is because this idea of high oleic soybeans is an old thing. Actually, our transgenic GMO versions of these, that various company sell that have gone through a regulatory process. So it's not like it's a new product class. It's just been done in a different way. But it's, again, I think still an important point tomorrow. Now, how did they accomplish this edit? Again, this is showing the process where oleic acid is converted by a dehydrogenase to linoic acid. And they're basically targeting the FAD2 gene, again, the different variations, the two different copies of it in the genome, and just basically mutating out its ability to catalyze this process. And so, you know, something that CRISPR-Cas9 could also do, but they, for intellectual property reasons, they have the right to talent. And so they performed it this way. Again, I presumably even started with CRISPR-Cas9. Anyway, so those to me, I think that's the CRISPR 2019 year review. I think to talk about it, it was an exciting year for, I think, the consumer slash patients in terms of these things. Entering the larger cultural and economic realities and frameworks we have on the planet. There are lots of new tools that I think are going to be interesting. And we'll see, again, right now, the things we target and the things we want to change are based on no mutations that occur and finding a way to do the same thing. And so I think the future will find maybe different ways of accomplishing those types of things. So I'm going to go back and look at the local chat for the recent questions. Thanks. I'm glad everyone enjoyed it. Listen, I'm going to find that there's one question I saw, and then I'll take other questions from local chat. Before we go on, let me thank Chantel and others for helping host this. Looking forward to a great 2020. Okay, so George had a question, George Newberry asked, is this also where a mitochondrial transplant might come in? Assuming I'm in the right part of biological science. Let me say there are attempts and ways to edit mitochondrial as well as chloroplast genomes. You know, I haven't seen a lot of stuff in terms of human patient therapies or our mitochondrial-based diseases that are out there that might be worthwhile to target. I'm not really very familiar with a lot of it, but I think it's feasible and there are some people working on it. And then there's another older question about, I think, genome mutations and variation. Like something about how do we know mutations or can we use Cas9 to discover stuff? Okay, whoever did that is going to have to repeat it because I've scrolled back and I'm missing a lot of conversation. All right, so George asks, might these CRISPR edits cause mutations in humans? And I presume you're talking about off-target mutations. And you know, I think the important thing to keep in mind is that people are showing that CRISPR Cas9, when engineered well and correctly, has very few off-target effects. And we always can track those down and discover them. And so, you know, it's a possible ability, but in terms of getting through therapies, regulatory frameworks, we have ways to help get away from that. Edgar asks, if you can have my slides, they will be posted to the Science Circle website. Shantel will put out, I think, links for that and let people know. Vic has a link to the Harvard Talk about CRISPR-29. A wired guide to CRISPR is out there in the local chat. So, Adriel asks a question, what is the relationship between CRISPR-Cas on the one hand and program cell death and microbial dormancy on the other hand? You know, the question, I think, is connecting things that I'm not sure are being actively investigated. I'm not sure they're, per se, relevant. One could use CRISPR-Cas to initiate program cell death. And I'm not sure what you mean by microbial dormancy. Maybe you can rephrase the question. Now, Arianna asks an interesting question, which is, is it possible to kill bad mosquitoes with genetic engineering? So, I did talk about that before. Last year, we talked about CRISPR, or maybe it was 2016 when I talked about this one. They're these things known as gene drives. And you can actually, there actually have been trials and people setting up trying to destroy mosquitoes by using gene drives where you get males that are born, but they're always infertile. So, you basically can induce a population crash because the few males that are born, the males, again, they're not the ones who bite you and transmit the disease. But they can crash populations by ultimately not having any offspring. And the few offspring that are born are males that are infertile, but then do breed and then waste the female mosquitoes' time. I think, and that's a very active area of research. Okay, so yeah, Vic is reposted in local chat. There's one question about joint DNA. And, you know, I'll just say it right here that James Watson was kind of an ass hat by saying that in the first place. Because on the, he was in essence trying to criticize Barbara McClintock's work on transposons and she was making the case that they were doing interesting and important and program things. Now she was not correct about the way they program gene expression. But the way we think about what we call mobile elements right now is that they are, in essence, parasitic. They're trying to, they're being selfish genes that are living and residing in other larger host organisms, trying to do their own thing. And, but what's interesting is that they end up becoming a very important source of genetic variability. Right. So when you hear someone like the Discovery Institute or Michael be he saying, oh, we can't explain evolution through point mutations. You just turn around and say to them, dude, there are lots of other ways to get mutations, including chromosome rearrangements and junk DNA and pseudo genes. And these are all things that you can use to tell them mobile DNA and say these are all parts of genetic variation. So there are lots of examples of what we call mobile elements that have ultimately become important for evolutionary adaptations. So I have to answer your question, Vic. You know, we'll see. So tagline just says, you know, messing with female mosquitoes and messing up maybe a food source for other organisms in the ecosystem. How bad is that people have actually studied that. So the fact is there are very few. Well, they hypothesize they're very few and they couldn't see this when they did the studies where any given predator mosquitoes was explicitly adapted to one type of mosquito. Remember malaria is only transmitted by an awful ease. And so eliminating a particular species of mosquitoes has is largely in consequential to larger ecosystem. So I think that that's an important point to make but people have studied because that people have raised that question before. I think that's an important one. Let's see. Watson was an asset or he wrote this. Oh, you're being sarcastic. Yeah, you've read about him too. Let's see. There was another question I think they had which were George. Oh, yeah, this was the question I was trying to find. Do we know which recessive traits that can be turned off that might not otherwise come in handy in future evolution. And so this is the other kind of more basic research area. That, you know, it's it's foundational. It's not directly trying to lead to therapies. But the basic idea is that in the lab. You can use cast 9 to do lots of mutagenesis. Random mutagenesis, semi targeted mutagenesis, make model versions of gene. Understand stuff. That's not already known. Right. So discovery phase. So, I think that's an important use of this. It's not something that I would say was a big highlight. There wasn't anything that came out of that this year. That's really big. But, you know, the idea of using base editors or CRISPR cast 9 to create genetic variation and then just see what you get out of it. You know, in model organisms is a big area that is going on. Is that a question for me day about the dinosaurs. Yeah, I think, I think we'll probably never really be able to do that. Unless we can actually get a sequence of an ancient dinosaurs DNA. Oh, so Berrigan, I think is pointing out I use a jargon term. Accidentally, which is a model organism. And the idea of that is, you know, humans, there's lots of complications in terms of doing experiments on them. As a whole organism in particular, they're expensive. There's review. There's ethics. So, but however, because we share ancestry, we share genes and in a lot of different ways that things like C elegans and nematode fruit flies mice. Even yeast, our model organisms that we understand how gene function works in those. And then we can use that to translate into how human genes work as well. So yeah, thanks for catching me on that one. Yes, even Z flies. So, oh, actually, like in the plant world, you know, there's some people use switch grass, because it's easier to work with than say corn or rice or wheat. And then you can try and translate what you learn from an organism into the actual product. Oh, okay. So George is refocusing his question, which was my mutation question was in regards to seeing vegetation that received gene editing. Again, the nice thing about most plant editing, as compared to say human cell therapies and trials is it's very feasible to. So what the study from young at all was showing, you can do the edit you want in a plant and you can actually to some degree be as messy as you want. You can actually have extra stuff insert in the genome. You can have extra edits, but with plants you can always back cross it and that's what you almost always do you back cross it several times, and then do an analysis to make sure that extra stuff for mutations did not occur. So anything that go even though, in a sense, gene edited plants in the US regulatory scheme aren't considered GMOs in terms of those regulations. You still have to do the due diligence for any product you release to the market. That it is what you say it is. And so the idea of we edited this but it didn't make other. We didn't create other things or leave, you know, some DNA in here and other places that all still in effect. And that is something any good company would keep track of anyway. Okay, so I hope that answers. Thanks for coming. Thanks for coming violet. Any other questions before we move on, I think I'm glad you enjoyed it. I hope it's a good summary. The slides will be available. And again, the thing about CRISPR so popular now that if there are some like minor questions you have their YouTube's their other resources, lots of good stuff. Okay, so maybe this is a good place to end. And actually there's a day also clarify the question. So let me actually let me go back to Dave's question. They'll ask what what do I predict next. So they wanted to just meant in terms of like reverse engineering or trying to get dinosaurs, the idea of trying to reverse engineer a bird, because we know that those are ancestors of dinosaurs to try and like regress it into a dinosaur. To me, the thing about genomes is that there's so many little parts of it that have to do with regulatory schemes, right humans only have 22,000 or so genes. And it's not how many genes we have it's when we turn them on when we turn them off maybe what splice variants we make. And that's really hard to understand from just looking at sequence. I'm trying to recreate the developmental process by reverse engineering a bird into a dinosaur means you have to get all those correct. So I think that that's for her now. If you can find enough mutations that say remove the beak that create teeth, you could in a sense, descend a dinosaur from a chicken. But that's not really, I think that the idea of going back and recreate dinosaurs. So let me finish, I'll finish. No more questions. I'll finish with this last good question, which is what next. And what do I predict maybe for the upcoming years. You know, I think clinical trials will be shown to be effective. And that's what Chris for cast night editing works. And that some of these will hit phase two. I think in particular for cancer immunotherapy there's going to be a lot of exciting, exciting therapies that happen with that. In terms of plants. We're expecting a lot more of these to get released and limited release. So I think one of the interesting mutations are benefits to farmers and consumers. You know, my long term prediction would be 30 or 40 years from now. The difference and the new types of products will have on the store shelves will be like the difference between the early 1900s to late 1900s. And so I think there's that's something to look forward to the problem with agriculture and livestock is that Europe has basically said we're going to regulate genome editing like it's regular. Genetically modified organisms. And so that is really slowing down the progress of what people are excited to try and do and release as a, you know, profit making products. You know, I think. But I think the fact that we have these other new tools, I think we might be seeing people explore more with trying to change gene expression profiles of things that have some degree of effect. You know, people who have kidney problems, happily go spend, I mean, not happily, but that's they are willing to go spend hours a week in a dialysis center in order to receive a treatment. And so the idea of having some sort of therapy that might be high cost. And you have to get over and over, but it is known to be a lot safer and it's not germline editing that might be a feasible pathway for certain types of things. Anyway, that being said, sorry, I can't be more specific, but it's really hard to predict the future. And I think that CRISPR cast nine with the complications of how we think about germline editing and the ethics of that and the regulatory schemes and agriculture and human trials, very complicated. Okay, so I think with that what I will do is close out my voice chat. And I might do a couple things in local chat, but otherwise I'm going to close out close out voice. Thank you all again for coming and your attention and listening. Hope you enjoyed it. If you have any questions, you know, just contact me later. And again, I want this to be an annual thing. So hopefully it'll be a CRISPR year review 2020 21 22 etc.