 So, just in terms of introduction as well, I am in real life Stephen Gager. I have done and published work in CRISPR, working for the company that's now known as Corteva AgroSciences, and here I am in second life as Stephen Zootfly. Now, one thing I want to just quickly mention is that of course there are lots of financial interests in CRISPR. I work for a company with substantial research and intellectual property, but I'm not here as a representative of the company and nothing I talk about really should be construed as investment advice. And I'm going to talk about one piece of published work against that proprietary that a colleague of mine published in the CRISPR area. And so the structure of my talk, again, now what I normally do for this talk is to give a lot of the updates of what the exciting stuff was in the year. And I will do some of that at the end, primarily talking about a few of the interesting new developments in a little bit of an update on what I talked about last year. But primarily I want to talk about the Nobel Prize and really again give this history of what were all the building blocks to their one discovery. Because I think it's important to recognize and the Nobel Prize community knows this too. We recognize one or a few researchers who made a substantial contribution, but very rarely is that done without the building blocks of many other researchers. So the award, again, technically the quotation described is for the development of a method for genome editing. But I think it's important to recognize that the reason this was awarded as compared to other methods of genome editing that have existed before is its versatility and its power and simplicity. And that I think is an important part of the award for Emmanuel Charpentier and Jennifer Doudna. So one of the main source, the basis of my talk is borrowing from a website, which I will throw into local chat really quickly, and actually I won't grab that later, is from the Broad Institute. And so the Broad Institute hires a lot of people. They have a substantial intellectual property position in CRISPR because Feng Zhang was awarded a priority patent, and that's a patent fight that's still going on. And so one thing I want to decide to do is to award, talk about the Nobel Prize, not get mired into the intellectual property fights that are going on with it. Now, but they do provide a very nice timeline. And what I've done is tried to go back and try, I'm going to try and give you, take you through a thought process of a couple of decades that led to Doudna and Charpentier's work. And so you'll notice on the left, there are key researchers, what year they did some work, and then I'll highlight the ones that are important. So what I have here, and this is work that people knew from the early 1990s actually, back when I was actually just a grad student, that when you sequence genomes of bacteria that you discover these little islands of repeats. And one of the features of these repeats is that they're a palindrome. So I hope I've tried to illustrate this on the slide by showing the word race car. That race car is the same going forward and going back. And if you actually even flip it upside down, it's also a palindrome that way. And what the term CRISPR stands for is clustered, regularly interspaced, short palindromic repeats. And so this was a feature of genomes. And at the time, people had no idea why they were there. And but what they did observe in these was that in between these palindromes were different sequences. And there really wasn't a clear picture as to what those sequences were, until we got to some more work by several groups, largely in Europe, where what they noticed, and this is Francisco Mohica, who was working in Spain. Although there was three papers published at the same time, where they did a more careful analysis of these sequences that were between the arrays, between the palindromes. And what they noticed was that when they compared that sequence to other sequences, again, one of the important technology things you have to have for this analysis to work is lots of sequences. So it makes a lot of sense that it wasn't until say the mid 2000s that you would have enough to perform, to even be able to perform and get a good grip on what this analysis was. And so I have highlighted here just one table from Mohica paper where they're just demonstrating that when they look at the sequences of these intervening sequences, a lot of them correspond to viruses, to bacteriophage, that infect bacteria, as well as something else known as conjugative plasmids. And plasmids are these little DNA elements and when conjugative means that they can transfer from one bacterial cell to another. And so there's stuff that carries on them. But it has to do with transferring about DNA coming from outside the cell that again might be very injurious to the cell. And so what these groups proposed was that somehow this could be some sort of bacterial defense system. There are other systems that were known for a long time. If anybody's heard of restriction enzymes, we have ways to break DNA. There are enzymes that do this from bacteria that's been known for a long time. And so that was the hypothesis. Now, that work though and how that would actually work wasn't very clear. I mean, so you can see this, you can see the representation and you have a hypothesis, but what that meant for immunity was not clear. Some of the early ideas had to do with interfering with RNA, which bacteria you, sorry, which bacteriophage used to do stuff. And that would be based on the precedent of I think the 2002 Nobel Prize was for RNA interference to fire in mellow. And that's also, again, another bacterial defense mechanism. But there was work that came along from Alexander Bulletin, again, also in France, where when they actually, they found an unusual array compared to what they've seen before. And what I have in this diagram is you see all those little black hash marks. And that's the example of the array, that what they notice these repeating sequences. But that big gray arrow over there to the left was the first time someone had seen a large protein with particular characteristics near those sequences. And they called it cast five. It's really what we now, in essence, think of as cast nine. But there were particular features about the sequence of that protein. And I have this written here in yellow, and this is a quote from the paper. Where it belongs, it has something that was an HNH motif. And that's something present in nucleases. And I have it here highlighted in a bolded, that it makes double-strand breaks. And so the idea here is that having a protein that is near particular sequences, maybe those are working together to make breaks in very particular, very particular places. And so this, again, and this is another step-wise in trying to understand how these things might work and why they're present in the cell. The next step in the timeline was this hypothesis based on a huge amount of work from Marikova, working with Eugene Coonan, to look at the phylogeny of a lot of these sequences coming out, and to look at these interspacer sequences, and, again, to make this hypothesis of why they're present. And so they did a much more thorough analysis of these target sequences, these homologous sequences, and saying, what are these really acting on, what are these trying to do? And if you look at this diagram, there's a lot of information here. But there's really just two key pieces, which is, one, these arrays of sequences with their intervening sequences are being expressed and then chopped up and then being used to target similar sequence to destroy it. And so, again, this idea of a bacterial immune system that is actually taking sequences from their target, that, again, if you think about the human immune response, we get infected. And then we have this ability to recognize them upon a repeat infection. This is known as immunological memory. And so somehow, this combination of targeting, defending, and then creating these DNA sequences derived from viruses is a way of having immunological memory. And the actual experimental demonstration of this came from, of all places, basically, the Horvath group, which was very interested in yogurt, essentially working for Dinesco, where they were trying to, again, the practical application of immunity is that bacteria, which are used to make gigantic vats of yogurt, can sometimes get infected by phage and basically spoil your food making process. And when that happens, that's a problem for what you're trying to sell. And so they were very interested in how immunological memory and the defense from infection works for bacteria. And they actually performed this demonstration of showing that the combination of this CAS protein that's nearby the array, the very particular sequences that are specific to the viruses that are infecting actually provide that specific defense. And so, again, this is just a basic bacterial immunological system to help them survive bacteriophage infections, just like our immune memory is, just like our immune system as well, from a basic concept of recognize, destroy, and then remember for the next time. Now, what's important, of course, in science is to really try and understand the details of how this works, right? So this is a phenomenological thing you can look at. But of course, from a science point of view, we want to be able to manipulate it. So another key part of this was understanding how the interaction of the proteins, these CAS, again, CRISPR-associated proteins, interact with these arrays. And this was work from John Vander Oost, again, in the Netherlands, where what they were able to demonstrate, again, this is only 2008, this is only 12 years ago, that these sequences that are coding for viral particles are able to be expressed and the proteins process them. So now what you have is this combination of a protein and these RNA sequences that are targeted to the particular ones. So I am now seeing a question from Syzergy, which is why would the phage sequences be next to palindromic sequences? And this slide is actually a really good way of identifying this, is that the palindrome, when you think about DNA, it actually folds up upon itself. So if you look at this picture where you see the ladder and the loop at the top, that's an RNA structure, which comes together in that structure because the sequences are palindromic. They basically pair with themselves. And so it's a combination of a sequence and a structure that the proteins recognize in the RNA in order to bind. And so hopefully that adds a little more. So I'm actually going to talk a little bit more about that in the upcoming slides, but that's now is a good time to recognize what the slides are trying to show you. So the next step, though, was actually really understanding how these sequences, what the protein is really doing. And so this is work, again, Marafini and Sondheimer, where their experimental system was to ask, what does the protein really recognize? Because based on the precedent of RNA interference, RNA interference is something that recognizes RNA and targets it for destruction as an immune response. But they really want to ask, these nucleus domains tend to actually recognize DNA. And so what they actually showed, this is just the text. I don't have a good way of showing this in a diagram, is that the CRISPR spacer was recognizing a gene present in other conjugated plasmids. But they showed through a very clever technique that the cash protein is targeting and breaking up the DNA. So it's not an RNAs and mechanism. It's actually a DNA targeting mechanism. So again, what's important, again, the thing to recognize that's important downstream for that is that targeting DNA is how you change heredity in organisms like ourselves, although RNA would, of course, be useful as a defense mechanism. And so, and I think that's an important stepping stone to then the next part, which is, we can work done by Moineau in Canada, again, trying to really cleverly understand and show why the spacers and the DNA targeting how they work. And so this diagram here, again, shown highlighted in the magenta, is again, you're probably looking at saying, Steven, more boxes with blobs in them. But a reminder that this is how a lot of molecular biology gets done is you take DNA and you put it through a gel in an electrophoretic field, and you can basically separate them based on size. And so the stuff that's at the smaller part of the picture are smaller. And again, you can use this as a representation for something being broken or modified some way by an enzyme. And that's what's important to show in this diagram. And the key point is if you look over here, as I bring my arrow down, is that in this part of the diagram, that big blob, the fact that you have two blobs on the left that becomes one big blob on the right is showing that the DNA is being cut, basically at about 100% efficiency. And what was additionally being shown by their work is that the site of the cut is represented by the sequence of the spacer. So this DNA that is being used to target this little bit of RNA that's being expressed between the palindromes is specifically being used to target where the target sequence gets cleaved. And so this, again, is telling us that there is, again, very good specificity and programmability to this type of platform. And so that really is what becomes the next body of work is not just noticing that bacteria can do these things, but really trying to understand and modify the system now that we understand what the platform is really doing. And so work that was done by, again, directly by Emmanuel Charpentier was a big, was a precedent to this, though, because what she noticed, again, using the ability to sequence small chunks of RNA and again, when we think about sequencing technology, this is an experiment that really would not have been possible probably before the year 2000, which is you can take bulk amounts of DNA, chop them up and then sequence them and understand the prevalence of them within a population. And so what I'm representing here from the Charpentier paper, again, highlighted by the magenta, is that these little gray boxes here, sorry, let me go this part first. So based on the diagrams that you've seen before, these little black and green shapes represent that palindrome array, the CRISPR array. And then these little arrows with boxes represent these CRISPR array associated proteins, the CAS proteins, as we call them. But then what she noticed for the first time was that to the left of those proteins was another RNA species. And so this RNA species, which she called a tracer, was something that was associated with these arrays and she also demonstrated was necessary for cutting activity. Now, the same time as this work, the Cisgnus lab in Vilnius, Lithuania, Vilnius University in Vilnius, Lithuania was demonstrating similar work that this CAS protein is a nuclease and that when you mutate the nuclease, it doesn't work as well. So again, when you look at these blobs here, which I'll put the arrow by, the wild type protein, the normal protein, gives you these smaller, these extra bands, whereas when you mutate it, you get many fewer of those extra bands and also show that when you bring these two pieces of RNA together, you can get cutting and that what they're really representing in the way the protein works is that the spacer basically goes and pairs with the target DNA. The CAS protein is opening it up into this loop. So you notice the sequence that's in red, that's the DNA that you normally know is double-stranded, but in fact is being opened up and then the RNA spacer comes in along with the protein and then this nuclease activity comes in and makes two cleavages so that you're making a double-strand break, not just making a nick, but actually breaking both strands of the DNA. Something I haven't talked too much about is the idea of a PAM. And this PAM is an important part of the sequence that you need for target recognition, although it's actually not a part of the RNA. And so the final step in the CRISPR discovery, the CRISPR timeline, is what we have next coming from Charpentier and Dowden. So one thing to keep, that's interesting about this is that Charpentier was very interested in the basic biology of these RNA species. What is this array doing? And Dowden was actually not involved with this field at all. She actually was someone who really studied RNA and how RNA processing and enzymatics worked. She was not involved in the CRISPR. So the Charpentier, I don't know the history of how they actually came together, but then they decided to collaborate. And so this collaboration yielded this final step in the process that turned this into a very useful biotechnology tool. And what I have here in the top diagram is, again, work very similar to what you saw from the Cystmasus group, is that this protein has these DNA cleavage domains. When you mutate them, they don't work as well. So again, that's why when you look at these white blobs in the dark background, that under wild type, you see one band of one particular size. And then when you have the mutations or the double mutation, they change. You don't see that same DNA species. And they also showed that this is programmable and that if you change the sequence and you provide a target, then you get very specific target, you get very specific cleavage of that target. And so again, showing this programmability of the cleavage site. And the other final thing, and this is the thing to look at in this diagram, is that the work I've shown up to this point before is in this diagram here, there are two RNA species. There's both the tracer as well as the CRISPR RNA, the CR RNA, as well as the tracer RNA. And these are separate things. If you trace how these go together, they're not connected. However, and this is the work that I think made this much more efficient and much easier to work with, if you look at this diagram, you now see this purple loop, a linker loop, that connects the CRISPR to the tracer. And so now this is known in general as a guide RNA, a chimeric species that is able to do very specific programming as easy to make in the cell. And so this basically demonstrated the programmability, the versatility of this technique as all stuff that can work within the test tube at relatively normal conditions. So in summary, leading up to the Nobel Prize, and this is a diagram taken from the Nobel Prize website, that starting in the early 2000s, people had noticed a weird thing in bacteria. And through a bit of work, the ability to have lots of sequencing, they had this proposal that worked in for bacterial immunity. And then through a decade of molecular and biochemical characterization, ultimately this led to a tool that has this high programmability and versatility to cut DNA in particular sites. And so again, this is essentially what Dowd and Nsharp NTA were awarded for as being the first to demonstrate this in a programmable bacterial system. Although I think it's also important to note that the Vilnius University Group was working concurrently and they actually submitted their work before the Dowdna Group did. And so they were really working in parallel at around the same time. Phil has an interesting comment in local chat saying so much has been discovered since then. And I'm gonna talk about that next because I think the important part of what the Nobel Prize is trying to recognize is how this becomes a platform to do so much more. It's not just that they can cut DNA, but what does it mean? And the next slide that I'm gonna show, which really comes into this idea of what does this mean? What did the researchers think about the time? Was some very old work from the mid 1990s, actually, from the Maria Jayson Group that demonstrated that if you can make a double-strand break in an embryonic stem cell, then the ability to put the DNA you want into that spot increases 54. You go from not being able to put DNA where you want to sell to basically putting it at a relatively high frequency where you want it to be. And so this idea of having double-strand breaks being in human eukaryotic cells as a way to do genome engineering had been around for a long time. And in fact, there were other groups that developed enzymatic ways of doing this. And I'm not gonna talk about this. This is a talk probably for some other time, but this citation work shows how there were other tools for doing double-strand break creation in cells known as zinc-figured nucleases, talons and mega-nucleases that are fine tools and some of them are actually still being used for certain purposes. But then once CRISPR came along, the number of citations, the amount of work on it exploded exponentially and basically made the other ones in many ways largely obsolete. And so that's the important thing to recognize is that there was this basis for people understanding how to do genome engineering, how to engineer proteins and how double-strand breaks are important. But the versatility of this tool is something that basically made it that next generation to be able to do something useful and broadly applicable. And so the next slide, again, from a review when I showed something similar to this last year, is that now that you have this ability to guide a protein somewhere, you can make a double-strand break. And this is the classic way of thinking about it is that we can do basically add template DNA, which again can be a gene, can be other stuff you want and put it into this particular site in the cell. And so that of course was, again, the initial idea of how genome editing would work. But other groups have come along and done other really interesting things is that you can actually put mutator elements on CAS9. So if you actually take away CAS9's ability to make breaks and make it so it's only a nick, it's only cutting one strand of the DNA, not both strands. And then you add something known as a deaminase. The cell will come out long repressed. It's not breaking the DNA, but it will mutate a sequence. And again, what's demonstrated here is changing a G from a T. And the ability to do that, again, depending what you want to do with your target gene or how you want to modify an organism, that can actually be a very useful thing to do as well. And then one of the developments I talked about last year was this idea of taking small sequences of DNA and using reverse transcriptase, which is called prime editing, where now instead of just being able to mutate a sequence, you can actually put in some sort of targeted template amount of sequence. And again, this is something to talk about last year. So I'm not gonna belabor the point too much, but it's really an important idea that CRISPR now is this platform that allows you to do a large number of things based on this ability of easy programmability and targeting of an enzyme, where all you need to do is change an RNA sequence in order to get it to go where you want. And because it's an enzyme, you can attach other parts to it. And so, yeah, and I think some, what I see in the local chat is talking about, like you can buy CRISPR kits, you can do biohacking. You do not need a large laboratory to be able to do this. And I guess, you know, Phil asks, is there a danger to letting people do whatever they want with genome editing? I think the answer to that is yes. But I don't wanna go into particulars. I think, you know, that's, I'd rather talk about the promise of the technology as we think about it in the control of professional, medical, pharmaceutical, agriculture environment. So do I have any quick questions about the history of the Nobel and the research leading up to it? Think about that process of science before I talk about the advances this year. Syzergy mentions a natural selection, which is a Netflix documentary about CRISPR being used by amateurs. Again, I haven't watched that one. So I don't, I can't comment on that one too much. I'm just gonna pause to see if there are any other questions for the first part of the talk. Well, if there are no questions on that, I will move on to, I think, what were some interesting technology developments for the year. So one of the interesting aspects is that, of course, once you have this protein where you know the sequence and you can biochemically characterize it, then you can start trying to redesign it as well. And so one of the limitations, again, one of the bigger limitations to be able to use CRISPR-Cas9 in any way you want to, is the fact that you have this PAM sequence. And what the PAM sequence does, it's a very specific sequence next to the sequence that's being targeted. But for Cas9 or any individual Cas protein, it's a very specific sequence. And so that means if you're target, if the gene you wanna change is not near one of these PAM sequences, then you can't really target it very effectively. And so there's been a lot of work where people have tried to, again, either find alternative Cas proteins that use different PAMs. But this work, which came up this year in science, was actually taking all of the seek, the biochemical information known about the part of the protein that recognizes the PAM and trying to generalize it. And so that's what's being shown here in this diagram, is we know the structure of it. We know what this, the interaction part, what it does. And so they were essentially re-engineering the sequence of it. And again, what I wanted to show here over to the right, these little, what looked like building block type of toy, what it was called, ticker toy type shapes, is this is actually recognizing at a very fine scale what the actual structure and sequence of the protein is in this very small location. And so they actually know how it interacts because we have crystal structures, NMR structures that give us a very high resolution picture of what these proteins look like. And so the final result that they found, and again, you don't have to understand all the details of this experiment, but the basic idea that the wild type sequence primarily recognized with this one yellow bar, it works very efficiently with that one sequence that represented by the yellow. But then after they've modified it, these other sequences also become more efficiently used as well. And so the fact that you go from just the yellow bar to multiple bars being relatively large means that they've made it a much more generalized protein that can recognize more sequences in the genome. And so I think that's kind of one of the more interesting technology tools and advances that came about from CRISPR-Cas9 this year. I will mention that there have been lots of people trying to characterize lots and lots of different cast proteins that have just different PAM recognition sites. That's work that's been done at Corteva as well and published last year. And there are other non-Cas9 cast proteins, like Cas12, or some other ones that have slightly different properties and they're a little bit easier to work with as well. The next thing I wanna do, that is give an update on some of the clinical stuff I talked about last year. So some of the target genes that people really want to target in order to have an effective clinical outcome have to do with sickle cell anemia and beta thalassemia. And I don't wanna go through all the details with that, but you can basically replace people's not working well red blood cells with the fetal version. And there's actually a relatively easy trick where you delete a repressor and you basically express that whereas most people that are adults don't have fetal hemoglobin being expressed. And so they developed a treatment for this. And actually what's interesting is that they're still only in phase one trials. And as a reminder, phase one trials are just safety. They're not efficacy treatments. That's where phase two comes in. But in a couple of the patients from this trial, being conducted by Vertex Pharmaceuticals and CRISPR Therapeutics, they actually are seeing promising therapeutic value to the people who are in the initial test group. Now there's another strategy. So cancer immunotherapy, again, it's a long, long standing therapy for cancer. One of the problems is that when you take cancer cells out of the body and re-inject them into a person, they have these inactivation switches. And unless you inactivate that inactivation switch, then they're not very effective as you take them out of the body and put them back in. And so there's a gene known as PD1 where instead of using a chemical inhibitor, they're actually trying to use CRISPR to edit out its expression in these T cells that are taken out and then put back into the patient. The only thing that really occurred in 2020 in terms of that trial is that as far as they can tell, they're passing the safety barriers. There's not any particular clear efficacy change in how well they're working. The last one, and again, one of the therapeutic targets that people almost always go to for new technologies is the eye. And the reason for that is the eye doesn't have exactly the same immune response for a lot of cells you might target and that it's actually a relatively easy way to deliver exogenous materials into a particular organ. And so there's this Lieber congenital amaurosis, which is, which are characterized by these mutations in a receptor where it's accidentally being misplaced. And I begin, you can go back and look at my slide from last year, but that when you can basically go in and edit out that mutation and then the normal gene, the normal protein gets expressed and makes a normal protein and you can actually recover the ability eye to work. Again, this is not a developmental structural problem with the eye, it's just the expression of one protein. And so they're actually seen in this particular trial. Again, this is the first direct injection, direct trial of CRISPR going into people and in fact it's showing efficacy that's helping with their eyeballs that they're actually able to see again. And there's actually kind of an interesting twist to the story where they tried to do it where they injected it in just one eye and let the other eye be the control. But in fact, the way this injection works is that it seemed to also carry over to the other eye. And so again, these people actually are getting again, a permanent fix to their eye problem because you don't need to go back and keep re-editing. Once you've edited this in enough cells and get that gene expressed correctly, then it's just a fix. You're actually are fixing the disease. So Phil, yeah, Phil, this was the topic I debated whether to bring up, which is cause one thing that did happen in 2020 was there was the second HIV-treated person who's now basically considered cleared of HIV that all of the traces of the virus have been gone from him over the period of decades. And so, but that was true like retroviral type of work or sorry, the combination retroviral work but also a stem cell transplant. And so there's been work cause we know the gene that can provide that if you are missing the CCR5 gene, HIV can infect your T cells. And so there have been people who have attempted to modify a patient's T cells to mutate that CCR5 and then make their cells resistant to getting another round of HIV infection and then they get their immune system back. Those have so far have not yielded any more cures. Although one thing that if, as I look through some of those data, I don't know that they cured the people of their original stem cells. So when you get a stem cell transplant, you actually get all of your normal cells eliminated and then you get the transplant. And I don't know that they were doing that with the CRISPR-based trials. So, but again, that's another very interesting area that if we know the targets of viruses, then we can modify our cells in a way to become resistant to viruses as well. All right, so here's another really interesting story and this is the one that again partly comes out of Corteva but also was worked on by Pukta over in Germany where if you look at this top left slide, what I'm trying to represent is what you see, what you see are a bunch of parallel lines that are lots of different colors. And this is genomic sequencing representing chromosomes, a chromosome in corn. And what you'll notice if you look at the middle of that slide, there are a couple of these where the line is not parallel. Basically it's skewed. It's rotated 180 degrees. And what's interesting, what that actually represents is the fact that some part of the chromosome got turned around. And this is something known as inversions where again, through random normal processes of evolution, chromosomes sometimes undergo inversions. Now, the problem with inversions is that they don't breed well. And so what these different colors lines represent are actually different corn inbred lines, which are used to make the high yield hybrids that basically are the underpinning of our corn yield revolution since the 1920s. And so, but the problem is if you have some of these inbreds with these inversions, they don't breed well with the other corn. Now, from a company standpoint, if we have a valuable inbred, we'd like to be able to fix that. And so this was worked on by Sergei Satavish, a colleague of mine, where they use CRISPR, and this is represented by the scissors in the diagram to the right, where they use CRISPR to basically try and cut at those inversion points, and then hope that the cell rearranges the chromosome in a way that it swaps the parts out. And so on the far right, where you see the blue on the left and the orange on the right, that's basically making the original chromosome back to the way it was. And so then you have an inbred that works. In fact, they got it to work. And so that was pretty amazing. And something that is useful for being able to make and have cross compatibility different inbreds, which is again, like I said, an underpinning of our agricultural revolution. Now, again, what they get published first was when I show down here in the bottom hand diagram, is that in Arabidopsis, or again, the mustard plant, again, a very common model organism for agricultural science, that they also show this inversion, where if you look at this chromosome four, on the top chromosome, it's gray and then yellow, but on the bottom chromosome, it's green, and then the yellow split up. And again, that's the representation of an inversion. And they also were able to go in, make breaks with CRISPR and basically put them back and tacked in the way they originally were. Now, what's important to recognize, is that once you start making breaks in DNA, and this is a bit of the cautionary tale for this year, several groups at the same time show that editing or making double-strand breaks in human embryos leads to things you don't want. And so, what we have in these three different examples, and they're all pretty similar, what they were trying to do is, if you have a gene that you know is defective from one parent, coming either from the sperm or the egg, if you were to make a double-strand break in the demutated version of the gene, then it could be repaired by the intact version that's coming from the other parent. And that's a good strategy. In fact, it works about 50% of the time, but as demonstrated by this, again, as demonstrated in the diagram with lots of data behind it, about 50% of the time, the broken chromosome will either delete sequences or sometimes the entire chromosome itself will just disappear. And so, what this represents is what is still a fidelity issue with how CRISPR-Cas9 works when it comes to human gene cell editing. And I think that a lot of you know there was the very beginning of the year the Chinese scientist was jailed, hey, for having performed unauthorized CRISPR-Cas9 experiments in several embryos in an attempt to make them resistant to HIV, which their father had. And so, this is the type of work that says, hey, you know, you gotta be a little bit careful with exactly how this works and how this happens before you really start experimenting in people. But again, it's one of these things like I mentioned in the previous slide, one of the nice things in the agricultural industry or other, say, bacterial or genome engineering areas, you know, if something goes wrong in some examples of it, you just recognize it and then you don't use those materials anymore, you just pick the ones that worked the way you wanted to. And then the last part, the last kind of thing that was new in 2020, and one thing I haven't mentioned too much is how CRISPR-Cas9 systems can be used to detect sequences, not just to break them. And so, this is work that came out. So people have known for a long time that you can take out the DNA breaking parts of the cas proteins and the RNA, sorry, the target sequence recognition part still works. And so, some work that was, so what hit the headlines this year was at this group, this company called Sherlock, developed a technology called Sherlock, which is an acronym for basically recognizing and targeting DNA. And it basically came out with a COVID diagnostic kit. And what I'll just mention again, this is another one of these diagrams where you have to have a lot of knowledge to really understand it, but the key point is diagram is that if you have some target sequence, again, you have a tissue sample from a patient, a saliva or blood sample, you basically do some amplification, you create lots of the DNA, you do lots of enzymology, then you mix that with the protein, your CAS protein, in this case, CAS 13, which unlike CAS 9, really naturally recognizes RNA, not DNA. And then through using those fluorescent tags, you can basically very quickly visually identify the presence of a sample or not. And so this is one of these things where if you know the sequence of a virus that you're interested in, you basically program this to recognize whether that target sequence is present in the tissue sample. And so this is work done by several people, but I mentioned specifically the Sherlock group because they basically came up with a test kit right away. There's another group using CAS 12, which I have mentioned down here at the bottom, where they basically made a 20 minute kit. Basically, it's one of these things off the shelf, you can basically incubate a tissue sample with it and have a visual indicator. It's not like a pregnancy test. Within about 20 minutes, you can know whether the sample is positive or not for COVID-19. So that last group was also an interesting one. But this is the type of thing that once you have a platform that does something that's very powerful, you can use it for lots of different applications. So again, Baradon mentions another interesting target point. A lot of people would consider a mutation that leads to blindness or deafness as something that you'd wanna fix. But of course, the idea behind many these communities too is that people adapt and basically don't consider that a disability. So in terms of how the communities do or don't respond to those things, I don't know. But I know from the biological point of view, you kinda look at something as a scientist and wanna fix it. And again, that's something that, again, as we think about generations of people, potentially being therapeutic patients for this, it's not just the people that you have now, it's the people you have in the future. So I'm going to end there. I thought it was another interesting year. I don't think there was many technology developments or clinical developments as there were in 2019. But of course, the big news of it being recognized by the Nobel Prize Committee and being something that is something that, I think people immediately recognize how powerful the technology was gonna be, how much it was gonna influence science, culture, technology. That's what it is. So it's very interesting year for that. And so I'll also make a quick plug that if you wanna just have a conversation, a colloquial conversation on this topic, on Friday night at five o'clock, we'll have the first fireside chat. And so it'll be here on the SIM, it'll be invoice and again, come and basically you drive the conversation. I'm here and happy to answer your questions. So thank you all for your attention today. Thank you for coming and I'm here to answer any questions. Yeah, I will have the slides sent off to Chantel. I got a little bit behind on all the citations I had for this. So look for that eventually on the website and the YouTube page. Yeah, I mean, if you wanna think about Monday night, some of the interesting topics, which I purposely bypassed for this talk was thinking about the patents, the intellectual property field, the ethics. Again, I have presented ethics panels so I didn't wanna get too much into that. There's not really anything new notable in the world about it, but yeah. So Shiloh asked an interesting question, which is, are there such things as heritage corn plants and can CRISPR improve its survivability? Well, I guess in a sense, I mean, the term heritage, as I know it more through turkeys, like heritage turkeys, are the original things we find maybe in the wild or maybe only from older civilizations that were adapted for agriculture but are not being modified, although there may be some degree of breeding involved. And of course, what's important to think about largely in those terms is that heritage plants are adapted to an environment and its climate and availability for survival over a long period of time. Then once we start breeding something for agriculture, we start optimizing for more yield, maybe higher growth density, any number of things where we can lose some of those heritage traits that are useful. In a sense, yes. I mean, I think one thing that a lot of companies are doing is trying to catalog and maintain seed stocks that are unmodified. And a lot of it comes down to, can you identify the genes that are important or not? Yeah, you guys, and I think talked about before this like strange tall growing corn plant that has mucus in its roots. And the mucus is there to help it do nitrogen fixation. So if we could find a way to eliminate the need for nitrogen fertilizer because the corn have their own mucus roots, that'd be amazing. So those are the types of things. The associated issue with that always though, Shiloh, and this is true of the corn mucus corn plants is that that intellectual property is I think owned by the Mars Corporation. And so, you know, then we get into to those issues. Yes, Mr. G, it's nitrogenated snot. But it's what it actually is. It's actually an association of particular bacteria. And so this gets into the microbiome aspect of any number of organisms have these associated ecosystems that may have co-evolved. Oh, no, sorry. Yeah, so Shiloh, let me just be clear. It is a natural plant, but you still can patent the application of a gene into products for other people. So you can basically own the ability to use it in products that would go to the market. Yeah, you can't own a sequence, but you can own applications or ways of modifying a sequence within our patents. Again, if you go back to, if you think about the BRCA-1, the BRCA-1 was the first test case of this that nobody owns the BRCA-1 patent. There's no such thing. But the ability to make diagnostic test kits for BRCA-1 is something you can patent, which is, of course, just based on the sequence of the BRCA-1. I hope that answers that question. Yeah, Phil, I think the idea of being able to do chlorophyll and have chloroplasts, that'd be pretty cool. But it's a one thing we don't have. I mean, one thing that leaves that plants have, of course, are leaves. And so even if we had chlorophyll in our bodies, we wouldn't necessarily have the same thin membrane, high surface area to volume ratio to actually make the chlorophyll very efficient. So we can just stick with eating. Well, yeah, so we are approximately 10 to the 13 cells and we are a host organism to about 10 to the 14 cells. So numerically, there are more of them than there are us. Yeah, I don't know a tagline. I think we might just have to stick with eating plants and getting chlorophyll and energy from them. Sumo asked a really interesting question, which is, what is the prevalence of bacteriophage? And I think people estimate there are approximately like a thousand fold more bacteriophage particles than there are bacteria on the planet. I will say the economist did a nice article on bacteriophage a few months ago. That might be a good reference, but, yeah, fudges are everywhere. And, but the very, I mean, the evolutionary history of fudges are very interesting too, right? One thing that we can, and people are trying to do, for example, is to treat various pathologies in humans that in fact are GI tract with a fudge instead of antibiotics. Why not just ingest a fudge that specifically kills that particular bacteria? And I think that that's an interesting idea that fudge are also an untapped reservoir of genetic diversity. In fact, there are some, what people have recognized recently is that there are CRISPR systems that are encoded on bacteriophage because they will use CRISPR arrays to try and fight the CRISPR arrays that are in bacteria as kind of like this warfare. So those are known as anti-crispers. Well, there are different types of anti-crispers, but one of them is actually to have a CRISPR array that attacks the CRISPR in your host bacteria. So that's an interesting topic too. All right, any other questions? I'm seeing it's now 11, and I'm happy to answer some more questions. And I mean, just to follow up on the idea of genetic diversity, that is something that people are tapping into is to look at all the genetic diversity of CRISPR systems and all the bacteria and the fact that we keep sequencing more and more genomes, mean we keep finding more and more interesting CRISPRs. One example, the original Cas9 protein is pretty large as an enzyme and as a coding sequence. So one of the things that people have been trying to do for a long time are find much smaller Cas effectors that are easier to fit on, say viral vectors for therapeutic delivery. So we'll probably have more, there's always more to talk about every year with CRISPR. All right, no other questions? Then I'm going to close off the voice and I'll stay around in tech chat for a little bit, but otherwise I'll clean up and I will again, thank Chantel for organizing and hosting the Science Circle as a whole for hosting the event and for all of your attention. And I hope all of you have a good day.