Re-writing the Code of Life: CRISPR Systems and Applications of Gene Editing

good evening everyone and welcome to this year's crew nyan lecture the crew lien lecture is one of the oldest annual lectures of the Royal Society and it's our most prestigious lecture in the life sciences it was conceived by William Crone who was one of the original Fellows of the Royal Society and among the papers left in his death in 1684 were plans to endow to lectureships one at the Royal Society and the other at the Royal College of Physicians his widow later bequeathed the means to carry out the scheme and indicated that the request was for the support of a lecture and illustrative experiment which sort of shows the times for the advancement of natural knowledge on local motion or conditionally of such subjects as is in the opinion of the president for the time being should be most useful in promoting the objects for which the Royal Society was instituted so that has been taken to mean significant work in the life sciences and the first crew nyan lecture was delivered in 1738 and the lectureship today is accompanied by a medal and a gift of 10,000 pounds which I'm told has been wired directly so the check is in the mail so this year's were really very fortunate to have in this year's crew nian lecturer professor Jennifer Doudna who is also a foreign member of the Royal Society for her outstanding structural functional studies of RNA and ribonucleoproteins and for elucidating the molecular mechanism of the CRISPR Cassadine system and developing it for genetic engineering so to tell you a little bit about Jennifer Jennifer Doudna graduated from Pomona College in California after having grown up in Hawaii and she then went to work with Jack szostak for her PhD at Harvard where she became interested in RNA catalysis and continuing that interest she went on to do a postdoc with Tom Cech at the University of Colorado in Boulder and that's where she began her studies on the group one intron ribosome and to understand it people had been it was clear that you would need a structure to really understand details of the catalytic mechanism but nothing bigger than tRNA had been crystallized in the previous 20 years and this even the construct that well I don't know what that text says but anyway you better just listen to me maybe it's my accent so anyway in even in order to identify construct she had to do a considerable amount of biochemistry to identify a construct that was both interesting and compact enough to crystallize and she got her initial crystals and then moved on to a faculty position at Yale University where he solved the structure of the p4 p6 domain of the group one intron and this was a big milestone for several reasons it was several times bigger than tRNA which was 20 years old at that time and and so it completely opened up new understanding of RNA folding and and tertiary interaction motifs it also shed light on how group one intron could take part in catalysis although that work you know came later another important advance that the this work made was phasing of large RNA structures and this is something many people don't realize but the compound that Jennifer and her team used osmium hexamine was actually then the basis for phasing much larger RNAs like the ribosome and so really it paved the way for breakthrough in the crystallography of the ribosome as well now at Yale and then at Berkeley she worked on many other important ribosomes on the mechanism of silencing on translation and its control and these were all areas at the forefront of RNA biology at some point she became interested in the CRISPR cast 9 system and I'm not going to tell you anything about it because it's the topic of her lecture but I will say it is a remarkable example of how in interest in a what might seem an esoteric topic you know bacterial immunity to phage his revolutionising not only biological research but potentially human health and the work has now revolutionized the field of gene and genome editing and as a result of her very distinguished career she has received many honors and awards including membership in the National Academy of Sciences and as I mentioned the foreign member of the Royal Society she's received among others the Gardner the Japan and the breakthrough prizes she was named one of the most hundred influential people by Time magazine as if all this weren't enough she's also founded several companies to use this technology to improve human health she's been active in the public sphere especially in discussion of ethics around genome editing and the sort of social and ethical implications of it and finally I should say she's the author of a very highly readable book called a crack and creation which explain you know really lays out her journey into this field so without further ado I've welcomed jennifer to give her lecture well good evening everyone it's a great honor for me to be here and I'm really delighted to see many friends and colleagues and of course to have that very generous introduction bye thank you Ramakrishnan one of the people who has inspired my career in structural biology and thinking about the function of RNA as you heard in the introduction I've had a long-standing fascination with RNA molecules and what they do in biology but the story I'm going to tell you tonight really has two different major players one of them is DNA sort of thinking about the structure of the double helix and how this molecule encodes the information necessary for life it's also honestly the way that I got excited about doing biochemistry back when I was a young student growing up on a little island in the Pacific Hawaii where I learned about the discovery of the double helical structure and that book that described that discovery by Jim Watson was the book that inspired me to start thinking about a career in a field that would allow me to discover molecular structures I was fascinated by the idea that somebody could do that kind of work and understand the structure of molecules and the other major player in the story tonight is is bacteria phage that infect bacteria so this is a cartoon showing a phage landing on the surface of a bacterial cell and like like all viral infections this involves injection of genetic material in this case DNA encoding the information to make a virus into the cell and it was through the the study of a process that cells use an RNA guided process to protect cells from this kind of infection that that it was possible to understand the molecules involved and recognize their potential to be useful for a very different purpose and that's really what I want to talk about tonight is is really the work done by our lab in collaboration with the lab of Emmanuel sharpen TA to figure out how bacteria fight off viral infections and in the process use enzymes that can be programmed for a very different purpose namely for genome editing and so what I'm gonna do tonight is I want to start out by telling you a little bit about CRISPR biology and how we got interested in this as you heard esoteric area of biology and then how our work to understand the molecular process involved led to harnessing of this technology as a genome engineering tool and then I'm gonna I'm gonna I hope you'll indulge me a little bit I want to tell you a bit about research that we've been doing in my lab over the past six years to understand the molecular basis for this process and then I'm going to at the towards the end of the talk I want to tell you I want to turn to the topic of applications of genome editing and and the exciting opportunities as well as I think the really fundamental challenges that we face in harnessing this tool going forward but let me start by describing CRISPR cast as a bacterial adaptive immune system so this the existence of these systems was unknown to science really probably until the late 1980s so all of the beautiful research done on bacterial genetics in the 50s 60s and 70s hadn't uncovered the existence of a way that bacteria fight viral infections that allows acquiring resistance to viruses and that was because these are largely not operable in the kinds of bacteria that scientists have studied in the lab they occur mostly in bacteria that are growing out in nature somewhere and so it was really through that kind of discovery by a few laboratories around the world that it became clear that bacteria can acquire the from viruses and use it to protect themselves from future infection and here's how it works and this is summer summarizing research from several different labs but what happens is that here's the the membrane of a bacterial cell and here's the inside of the cell and if this bacteria isn't infected by a virus which injects its genetic material if the bacterium has a CRISPR system it has a place in the genome where it can store small pieces of viral DNA and those pieces are stored in a very particular way this is a call to CRISPR locus where there are a series of DNA repeat elements shown by the black diamonds that flanked these unique bits of viral DNA shown by the colored boxes so it's a very distinctive pattern that was first recognized by computational biologists who are looking at bacterial DNA sequences and noticed these things they didn't know what they were doing at the time and these reside next door to a series of genes that encode CRISPR related proteins are cast for CRISPR associated these are cast genes that encode proteins that turn out to interact with these RNA transcripts that are generated from the CRISPR locus shown here initially as a precursor molecule that gets processed into small pieces of RNA that each include one of the virally derived sequences and when those combine with the cast proteins they form surveillance complexes that can search through the cell looking for pieces of DNA that match the letters in this twenty nucleotide stretch of the crispr RNA molecule so it's a really beautiful system and you might notice that by these colors that each of these proteins is programmed with a different RNA sequence that can recognize a different type of virus a different viral DNA and then once that recognition happens the proteins are able to use base pairing between the RNA and the DNA to capture that piece of nucleic acid and allow it to be cut by the caste protein and this leads ultimately to destruction of the viral nucleic acid and protection of the cell so it's a very interesting pathway and we were fascinated I was initially fascinated by this because of the role of these RNA guided proteins and trying to understand what that might tell us about the evolution of these sorts of processes more more broadly not just in bacteria but also in other kinds of cells potentially and so we started working on this and this one of the things that's very interesting about these these CRISPR caste systems is that they're very diverse in nature and this is a cartoon that shows an attempt by the lab of Eugene Koonin and his colleagues to categorize the CRISPR caste systems according to the types of proteins that are found encoded next to the CRISPR arrays and so each of these little boxes is a different caste gene and they're color-coded by what they do in the system and quite broadly one can separate these into two classes called class one and class two that are distinguished by the number and types of caste proteins that contribute to the pathway so that's the class one systems have multiple caste proteins that are involved in protecting cells using an RNA guided mechanism but down here are the class 2 systems each involved just a single large protein that has the ability to protect the cell in an RNA guided fashion and so when I met Emmanuelle Charpentier at a conference in 2011 she told me about research that her lab was doing on a bacterium that had this type of CRISPR system here with a single protein called Cass 9 that was implicated as an RNA protecting enzyme and at the time nobody knew the function of this protein and we thought it would be fascinating to figure out how it works and so that was really the question that we set out to address together was what is function of this of this fascinating enzyme cast line and so in research that was conducted in our two labs across oceans and different time zones by two very talented people Martin Janek postdoc in my lab at the time and christiansí a graduate student with emmanuel these guys figured out that casts nine is an amazing enzyme shown here in this cartoon as the blue blobby II structure that has the ability to hold on to double-stranded DNA at a place in the sequence matching the twenty letters in the crispr RNA molecule this molecule right here and when that interaction occurs the protein makes a cut in the DNA and it uses two separate active sites to generate a blunt double-stranded break so in bacteria that's a terrific way to cut the viral DNA and initiate the process of degradation now Martin and Chris trial in ski figured out two other important things about this enzyme one is that in nature it requires a second piece of RNA called tracer shown here in red that interacts with the end of the CRISPR molecule to provide a structural handle forecast nine binding so it's a really a dual RNA guided protein and they also figured out that this targeting mechanism requires not only a match to the crispr RNA sequence but it also requires that this site be next door to a small motif in the DNA called the pam motif which for this enzyme that we started working on is a GG dinucleotide so two g's and in the DNA and that turns out to be important as I'll explain shortly for the mechanism of DNA recognition now martin jean genet being a wonderful biochemist and he actually had originally done research in Kiyoshi no guy's lab here in the UK before coming to my lab to do his postdoctoral work he figured out that these two rnas could be linked together to make a single guide form of the sequence that would have the RNA targeting portion on one end and the handle forecast 9 binding on the other and the really exciting thing about this development was that we realized when Martin did experiments with this single guide RNA that it made the system trivial for programming caste 9 either in vitro so biochemically or in cells and the reason is that now this is a two component system with a single protein caste 9 and a single piece of RNA that could be programmed by changing this sequence in the RNA to cleave any desired DNA sequence and by using it in that in that way it was possible to introduce double-stranded breaks into the DNA of eukaryotic cells plant and animal cells that would trigger a DNA repair and this this sort of recognition you know is one of those one of those sort of really precious aha moments that we all live for in science where you're doing experiments in the lab and you don't quite know where it's going you're trying to answer a particular question and then when you see the data you realize that the data tell you something unexpected that you were or explain something about biology that you couldn't have imagined and for us that was the case here because what had been going on in other other fields at the you know sort of for the last couple of decades before we did this research was a lot of work on understanding how DNA is repaired in eukaryotic cells in plants and animals where double-stranded breaks rather than leading to a quick degradation of the DNA and cellular death typically are detected in the cell and repaired by pathways that include non-homologous end joining where the ends of the DNA are ligated back together sometimes with a very small change in the DNA sequence or by a process that involves homologous recombination where a piece of DNA that has a sequence matching the sequence of DNA that was cut can be integrate into the DNA during the repair process and many scientists appreciating this this process recognized that you could actually use this to trigger changes to the genomes of cells by by introducing a double-stranded break in a desired place in the genome and the big challenge was how do you do that not so easy and so there were a number of technologies that have been developed involving primarily programmable proteins that could be engineered to bind to particular DNA sequences and these could work very robustly for gene editing but they turned out to be difficult enough to deploy as a technology that it had not been widely adopted and the thing that's so exciting about CRISPR caste 9 is that this system is essentially very trivial to utilize by people that have molecular biology training and so it became a tool that was almost immediately adopted very widely for applications and all sorts of different kinds of cells I'm gonna show you one quick little video here this was a movie made by Janet iwasa to illustrate how we imagine this CRISPR caste 9 enzyme functioning in a genome editing context so here here we are zooming into a eukaryotic cell so the DNA is in the nucleus and it's highly compacted into chromatin so wrapped around these histone proteins and this programmed enzyme so the caste line protein with its guide RNA will search through the DNA to find a sequence that matches the sequence of the guide RNA when that match occurs this protein has the ability to unwind the DNA helix cut the two strands of the DNA and then hand those ends off to repair enzymes that will fix the DNA here with quite a bit of artistic license integrating a new piece of DNA sequence and amazingly this enzyme that evolved in bacteria and to our knowledge does not exist in eukaryotic cells nonetheless works very well when it's engineered to go into the nucleus like this and make double-stranded DNA breaks so I'm going to come to the some of the applications of this towards the end of the talk but I want to turn to now is to tell you a bit about the research that my own lab has been doing to understand how this actually works we've been sort of fascinated by this question of DNA recognition all the way from you know how does this enzyme find a DNA sequence and and allow an RNA strand to inter sort of interfere with the double helix by opening up the DNA to form an rna-dna hybrid all the way to how does this actually work in the context of a living cell and I'll show you just tonight some of the experiments that we've done and data that we have that address these sorts of questions and I wanted to start by just pointing out that one of the really interesting things about caste 9 in this enzyme that emerged early on is that this is a protein that is a it's effectively a helicase it has the ability to melt apart the strands of DNA at a position matching the sequence of the guide RNA and how does that how does that work we've been trying to figure this out so this is a 3d printed model of a caste 9 guide RNA complex bound to a double-stranded DNA substrate it's a model that's actually based on an actual 3d crystallographic structure that was solved in Martin genex lab at the University of Zurich and what I'm showing you here is is the protein with one of its cleaving domains taken away so you can actually see the inside of the protein you can see that when it finds a matching sequence in DNA it forms an rna-dna hybrid inside the enzyme that displaces the second strand of DNA and in a process that we're still trying to figure out allows this enzyme to access a functional conformation that leads to DNA cutting and amazingly this is an enzyme that this without any external energy source so it's able to somehow coax apart the DNA double helix even in the context of a whole genome that's packaged into chromatin by melting apart the two strands but it doesn't use any external energy source to do that so it has no hydrolysis of ATP or GTP that drives this process somehow it happens independent of that and one of the clues to how this how this melting of DNA works has come from studies of crystal structures and looking at how the protein structure changes as it binds to guide RNA and then to DNA I'm going to show you one little movie here that just morphs between different crystallographic states of caste line to illustrate this process of structural change so this little movie starts with the structure of caste line alone and as it morphs to the structure bound to the guide RNA here in orange there was a you might have noticed a very large rotation of this part of the enzyme that actually creates a central channel in the protein where the RNA guide is located and then when the sin sign binds to DNA there's an additional change in the structure that accommodates that rna-dna hybrid and then finally there's a there's actually a sensor in this enzyme that detects the fact that it's bound to a complimentary piece of DNA and that triggers a final rotation of this active site called HNH that swings it into position to cut the DNA so this part of the enzyme contains the cleaving residues that actually generate one of the cuts in the target strand of the DNA now that final rotation that I just showed you and this in this video was not observed in the original crystallographic structures of the caste line because it turns out that it only happens when this protein is engaged with a bona fide double-stranded DNA substrate and that was possible to trap and and solve structures of by two people in the lab foo gojong and David Taylor in collaboration with the lab of Evan o galas and the structure I'll show you here it was obtained using a combination of crystallography and electron microscopy and I just want to show you what this looks like so here we are zooming in to the interior of the enzyme you can see the orange RNA with the DNA hybridized to it and here's the other strand of DNA and magenta sort of opened up inside the enzyme and that structure actually positions the enzyme to put its active sites in place to actually cut DNA and you'll you'll see coming into place here the HNH domain this time in green so you can see it rotating into position so now can it's actually placed properly to generate a break in this strand of the DNA and and importantly this requires recognition of this Pam motif but sitting right here next to the targeted sequence which for this enzyme is a GG dinucleotide and we understand now from crystal structures and and other kinds of experiments that there's a binding site in the enzyme that interacts with those G's in the in the DNA and somehow triggers melting of those first few base pairs that allows the start of DNA hybridization and eventually allows this entire strand to interact with DNA so it's really an interesting enzyme that has the ability to be programmed to interact precisely with DNA and also to sense whether it's on a bonafide target or whether it's on a not quite perfect matched DNA which leads to binding but no cutting of the DNA so I wanted to share with you a few experiments that we've been working on over the years to sort of address this question which is how does cast-iron find target sites within the vast excess of non target DNA and the first set of experiments that we did to address this was a was a terrific collaboration with the lab of Eric green at Columbia so Eric had come to Berkeley and talked about work he was doing you DNA curtains which are single molecules of DNA that can be used be sort of attached to a slide and then one can monitor binding and binding behavior of proteins that interact with the DNA by putting a fluorescent label on the DNA and using TIRF microscopy and this is what these experiments look like so each of these strings sort of green strands that you see here is a phage lambda DNA molecule so it's a 48 killer base pair piece of DNA these are each attached on one end to a slide and then they're extended when there's a buffer flowing across the slide from top to bottom and then in this experiment what we're doing is labeling casts nine protein with a fluorophore and then we can program that enzyme with a particular piece of guide RNA that will direct it to a site within the the phage DNA and what you can see here is that these enzymes are all lining up at roughly the place where we expect them to according to the position of the guide RNA binding and so it was by doing these sorts of experiments and these were conducted by Sam Sternberg in my lab and SCI reading a student in Eric greens lab that it was possible to do experiments that established first of all that you could get accurate targeting this in this sort of an assay and you can see very nicely the way that these proteins would line up on the DNA according to the position of their guide RNA and it was also possible to do things like measure the the binding kinetics and how long it took for an enzyme to find its target site in the context of this 48 KB piece of DNA and to summarize what we learned in those experiments we figured out that first of all there's very high affinity product binding by the enzyme that means that this is a protein that binds to DNA and cuts it and then at least in these sorts of experiments never let's go at least in the context of the time course of these these types of assays so there was no substrate turnover for each enzyme that we added to these reactions we would get one molecule of DNA cleaved so that argues that in cells there's probably a you know a lot of other enzymes or proteins that have to interact with DNA to displace cast line secondly we found out that binding occurs first that these Pam motifs about at least one of the ways that this enzyme operates is by searching for these GG die nucleotides in a sequence of DNA and slowing down only upon encountering those and of course they would be quite abundant in a typical genome but nonetheless this provides probably the first constraint for this enzyme to localize to places where there could be an actual target sequence for DNA recognition and then finally we figured out that target binding somehow triggers cast nine catalytic activity so this is an enzyme that is poised to bind to DNA but only once it's engaged with an actually a piece of DNA that has a match to the RNA guide is it triggered to activate and and cut the DNA that's probably important for survival of bacteria they wouldn't want to have an enzyme let loose in the genome that's making inaccurate breaks and potentially leading to cellular death so with those sort of observations in hand we said about doing a series of experiments that involved using dye molecules on the surface of the cast nine protein to monitor conformational changes we were really intrigued by the possibility from these comparisons of crystal structures that this enzyme might undergo a big structural rearrangement upon DNA binding but you know until you actually detect that and solution really want to be a little bit skeptical of you know was that really how it's working and so to test that this was originally the work of Sam Sternberg in the lab so what Sam did was to look at the various crystal structures of this enzyme and engineer into the protein sequence residues that could be that could be modified chemically with pairs of dye molecules in such a way that we could detect fluorescence resonance energy transfer or fret between the dyes and so what I'm showing you here is example where we have cast 9 protein you can see the difference in the inactive versus the active states of this enzyme and when we have dye molecules position here and here they're too far apart to give a fret signal when the protein is in the off state so not actively engaged with DNA but once this active site swings into position on a piece of DNA then they become very closely positioned and so Sam Sternberg originally did these experiments in bulk solution and got some very nice data that were consistent with the conformational changes detected by comparing crystal structures but we wanted to do experiments that would give us even more insight into the in behavior of individual molecules and this led to a great collaboration with the bio physicist on metal DS at Berkeley and his student Yahoo's and my student Janice so these these two students teamed up to tether cast 9 molecules to slide surfaces that were in a way that would allow them to do a single molecule fret measurements and in this type of experiment what was done was to use a piece of DNA that would base pair with the end of the guide RNA molecule in cast 9 and this protein of course has the dye molecules on the surface so we can monitor fret changes and then we flew into the into the system different pieces of DNA that have the pam site as well as a target sequence and the target can either be a perfect match to the guide RNA or contain different numbers of this matches and then we can monitor the fret changes that occur in cast line and I just want to show you a couple of the types of data that we got that started to really explain I think a lot about how this enzyme functions so in one of their experiments Yavuz and Janice were able to compare the fret signals that we observed for individual molecules as a function of the type of DNA that was added to the system and so what you're seeing here are and these are single molecule phred experiments so we're binning the front signal that we get from individual molecules of caste 9 according to the type of DNA that was added to the system and so when we have a perfectly matched piece of DNA that matches the crispr RNA these molecules show a very high fret signal and again that's consistent with these dye molecules being very close together so we're getting a very high high signal in that assay compared to what we see for RNA only here this is where we have no DNA added to the system and the molecules are all in a very low fret state so they're all in the off position and then what's interesting is what happens in between so when we have DNA molecules that have even a single base pair we start to see population of this intermediate state monitored by fret and you see that the more larger number of mismatches in the DNA the more we see population of that intermediate and with this and some additional experiments we were able to conclude that this represents a conformational checkpoint of caste line it's really a state that the enzyme has to pass through to become fully activated and it's really influenced by the degree of base pairing with the target DNA molecule furthermore we could do experiments of this nature where instead of tethering the cassadine protein one could actually just tether the substrate DNA molecule to the slide flow in the protein with its fluorescent labels and monitor the rate of change of the protein structure as it interacts with DNA and what you can see here at the bottom is that when we use a non target piece of DNA that is au has a perfect match to the guide RNA there's very rapid occupancy of that high fret State so very quickly within a few seconds this enzyme grabs onto the DNA and snaps into position to cut it whereas if we use a piece of DNA that has mismatches to the guide RNA we see that it takes a lot longer so these are ten to ten to twenty seconds here to even start to occupy that activated state and for the most part most of the molecules remain over here in this low fret inactive state again pointing out that this is an enzyme that has a sensor that is detect the degree of base pairing matches between the RNA and the DNA and then finally I wanted to show you that this activation is dependent on divalent metal ions so this is doing these same types of phred experiments where we have different types of divalent ions included in the reaction if we use no divalent ions and we add a key later to grab all of those ions and make them unavailable to the enzyme you can might see here that all of these enzymes are locked in this conformational intermediate state but as soon as we add just a pinch of magnesium 10 micromolar in this experiment we start to see population of that activated state and that grows in as we add different types of divalent ions but interestingly that's independent of the enzyme being able to actually cut DNA because if we use a version of caste 9 that has a mutation in the active site it nonetheless can occupy that activated state so it really suggests that binding and this conformational change are separate from actual DNA cutting which has been actually important for using this as a as a tool for DNA recognition in different types of genome manipulation experiments so just to summarize what I just told you this is a enzyme that goes through this conformational checkpoint that allows detection of base pairing between the DNA and the RNA and in data that I won't show you tonight and we were able to use this this understanding of the mechanism to make mutations in and figure out where this DNA sensor is in caste 9 and mutate it so that it makes the enzyme more accurate and even more sensitive to mismatches in DNA which is proving to be an important aspect of genome editing where we want to ensure that edits are made only at the desired position in a genome and not anywhere else so one other thing I wanted to share with you about the caste 9 mechanism is thinking about how we take this biochemical understanding and apply it to thinking about the way this enzyme operates in the context of a living cell and so from the work that we did with Eric greens lab using those DNA curtains we figured but figured out that the most our data were most consistent with a model where this enzyme rather than sliding along DNA to find a binding site is instead binding and releasing DNA very quickly and slowing down only at Pam sequences to allow initial base pairing between the adjacent sequence of DNA and this guide RNA and if there is complementarity there then this starts to form a structure that eventually looks like this if there's pole complementarity and leads to DNA cutting but how does this work in the context of a genome and so I had a wonderful student Spencer Knight who came to the lab a few years ago and he wanted to try to answer this question by thinking about how castellon would be able to interact with structures like this namely nucleosomes that are the components of chromatin in eukaryotic cells and so the questions were you know how does caste 9 deal with chromatin structure and how does it handle big genomes and how does it deal with nuclear organization sort of the higher order architecture of genomes and we certainly don't have answers to all of those questions yet but got some important initial information by conducting experiments in which we included a halo tag on the end of caste 9 and so this is actually doing experiments with the catalytically inactive form of the enzyme with a halo tag on the end so this allows coupling of a very small fluorescent dye to this protein and importantly one does this in the context of a living cell so by expressing this fusion protein in a living cell and then adding this dye molecule that will go into the cells and react with this halo domain over here we get a fluorescently labelled caste 9 and I'll just show you a couple of results using this in super resolution microscopy experiments to visualize the behavior of caste 9 and these are in living Mouse cells and so one thing that we noticed right away was that when we compared the kinetic behavior of caste 9 proteins that were programmed with a nonsense guide so they don't recognize a sequence in the genome compared to what we see forecast lines programmed with a guide RNA that recognizes an abundant sequence in the genome repetitive element called a sign we found that and you can probably just see it visually here that the these individual proteins moved a lot slower in the cell when they were programmed with a sign recognizing crispr RNA and you can plot this over here this is just the log of the diffusion coefficient plotted against the frequency number of particles with that behavior and so you can see that we get very rapid diffusion when there's no DNA recognition going on but there's a lot a lot of these particles have much slower kinetic behavior when they start binding to the DNA and so by doing these sorts of measurements it was possible to calculate at least roughly how long it takes for cast line to find target sequences in the cell and also to do experiments like this where we could actually visualize the behavior of these labeled caps nine proteins in the context of cells that were had an additional label in a protein that binds to heterochromatin these are parts of the genome that are really really compact and one question that we had was you know with this protein with its rapid kinetic behavior being able to get into these highly compacted parts of the genome you don't see much when I show it to you like this but when we compile all of these images what you can notice is that these bright regions are the heterochromatic parts of the genome and you can see that these colored paths show the the kinetic behavior of individual casts nine proteins much of the the time the protein is outside of heterochromatic regions but we see forays into these these structures as well and that's consistent with the observation that we can get gene editing in these heterochromatic parts of the DNA it just takes longer than it does in the rest of the genome so it's really a remarkable observation that this enzyme is able to somehow insinuate itself into even in these highly compacted portions of the genome which perhaps speaks to the dynamic nature of even these very compacted parts of a typical eukaryotic genome so in the last two minutes I just want to turn to the challenges that are ahead for taking this system and sort of the understanding that we and others are developing about how the function of this protein allows it to be a programmable enzyme that initiates genome editing and thinking about how we're gonna take it from where this technology stands today to solving real problems in clinical medicine in agriculture and in other areas of biology including synthetic biology and I wanted to point out a couple of things so I think first of all that when we think about challenges especially for therapeutic applications of genome editing I really see three major major ones one is delivery so how we can introduce these genome editing molecules into cells and tissues and patients how we control the way that DNA repair happens after the DNA is cut and and finally how we deal with the ethical challenges that come up when we think about certain types of applications of genome editing especially in humans so one of the things that that's happened over the last six years is just the remarkable acceleration of research in the biological sciences enabled by a number of technologies but including cash line and I just wanted to show you this plot so this is a I was writing a review for a journal on CRISPR Cass and I you know sat down on a Sunday morning to kind of go through PubMed and I told my husband oh this will probably take me a few hours to do this and I you know several hours later I was only not only gotten through two months of you know the first part of this year there were just so many publications and so right now this is probably now even out of date but about 8400 entries and this is just to give you a sense of what's happened since so really over the last six years where labs worldwide have started to employ gene editing to do all sorts of experiments and we now have a you know sort of a library of all of the different types of cells and organisms that have been edited using CRISPR cast line this is actually just a partial illustrated map here of these different types of organisms but it's really been remarkable to see the kinds of interesting biology that's been enabled by having a tool that allows easy manipulation of genomes and I just wanted to tell you about five areas where I think you know there's some really exciting applications that are happening and just give you examples of some of the things I think are most interesting in these different areas like there's lots of things I could have picked but these are some of the things that I've been thinking about recently and I want to mention research sort of fundamental research health care therapeutics agriculture and diagnostics so in in research I I'm just going to give you two two examples that illustrate the way that having a genome editor at your fingertips as a scientist has really opened the door to doing new biology so this was a picture that I got from cloudless blonde at New York University and his students who have been using CRISPR cast nine to to make genetic changes in butterflies that allow them to study in in genetic detail the wing patterns of these butterflies and this was something that until there was an easy way to edit genomes was possible to do only at the level of observation and just looking in nature at what kinds of Barents variant types of wing patterns would occur but now they have a tool that allows them to manipulate those genes exactly and ask questions that couldn't have been addressed in the past there was also a recent article this is actually from The Guardian talking about the work that Svante Paabo and his team are doing to understand the evolution of the brains of Homo sapiens and so they've been looking at the genetics of Neanderthals as some of you may know and experiments are doing currently are involved growing small organoids of neuronal cells in a dish where they can actually introduce into a human derived organoid a segment of DNA that's taken from the Neanderthal genome and they're focusing on three different genes that they think are important perhaps for the development of certain types of neural functions in Homo sapiens to ask whether they can recreate that in these settings in the laboratory again an experiment that would have been impossible to do without a tool that allows site-directed gene editing in terms of health care so there's lots of focus on biomedicine but one of the areas that I think is actually also very interesting it's thinking about ways that we might be able to have an impact on healthcare in the future that don't involve doing any gene editing on humans and that is this is an example here where George Church and others are interested in asking whether they can develop pigs that are have been genome edited in different ways to remove endogenous viruses and to make their organs more human-like that would allow these animals to be great organ donors for for human patients in our own lab we've been thinking about about how we might be able to employ the CRISPR cast nine enzyme to target disease genes and we've actually been working on a gene for Huntington's disease this is a well known gene that involves a mutation that was mapped actually back in the 1980s so we've known about this mutation but it's been impossible up until now to offer these patients any any anything really other than palliative care and so this is showing an experiment done by a postdoc in my lab Brett Stahl who was able to modify the Cassadine protein with its guide RNA so that this protein has cell penetrating properties and by injecting this into the brains of mice that are engineered genetically so that we can observe the editing reaction by simply watching the cells turn red when they get edited we can actually observe that we get really significant volumes of brain tissue that gets edited when we do these kinds of injections and we're actually working now towards developing this as a tool that will work in a larger animal model and in looking for even better ways to get penetration of these proteins so that we can edit enough cells that we would have a therapeutic benefit in principle in in patients and I want to point out that in agriculture there are just so many opportunities right now with gene editing I think people are very excited about what's going to be possible in the future this is a great example from the lab of Zack Lippmann at Cold Spring Harbor laboratories who was able to use CRISPR cast nine to generate strains of tomatoes that carry much heavier fruit yields than the unedited plants something that he told me would have been either difficult or impossible to do previously and certainly now can be done within a matter of weeks by engineering just the gene that is necessary to change for this very productive type of tomato plant to be produced and finally I just want to mention that in recent work by our lab and several others it's been possible to harness these CRISPR enzymes for detection of nucleic acid sequences and this is taking advantage of the properties of some calf proteins not cast 9 but other sort of related RNA guided enzymes to recognize DNA or RNA molecules and then trigger cutting of a fluorescently labelled piece of DNA or RNA and by doing this type of reaction it's possible to detect the presence of viral DNA even potentially DNA associated with cancer cells tumors in very small volumes and without using any kind of fancy equipment like a PCR machine so there's a lot of excitement right now about the potential to be able to detect DNA and RNA sequences using this simple kind of technology that might be applicable in a point-of-care setting and I just want to close by by circling back to sort of the some of the broader ethical issues and for me I think it was really back in 2014 you know sort of my evolution in you know in my own thinking and this in this whole field started with you know being incredibly excited about the potential of the technology and all the things that it was doing and all the papers that started getting published and people harnessing it for different applications to realize in quite quite that this technology was going to be could be used for different kinds of editing experiments and I wanted to just point out that fundamentally we can divide cell editing into somatic cell applications meaning non heritable changes that are made to DNA they not passed on to future generations versus what happens when we conduct germline genome editing where changes become heritable so they affect the entire organism and its future progeny and so this was being done from very early on and various kinds of animals and plants but you know it became clear quite quickly that this would work also in primates and people started to ask the question what about human germline editing would this be something that people should should do either for research purposes or for clinical applications and of course that quickly led to people imagining CRISPR babies and you know all the sorts of things that one might like to engineer into our kids but I think you know there is a very real possibility that in the future certainly not today but at some point in the future that this will become a reality it will become possible to introduce changes in a targeted way into human germ cells whether it's eggs or sperm or embryos that become part of the person and become part of their future lineage and that's a really profound thought if you think about it because it really means that we now have a tool that allows us to control our own evolution if we want to and the question is what do we do with a technology like this how do we how do we manage it how do we handle it I don't know the answer to that question but it did seem very clear to me that it needs to be publicly discussed and so together with a number of colleagues many of whom are in this room we got together and started talking about this and there was an international summit held on this topic back in 2015 and another one to be held this year in November in in Hong Kong and the first meeting actually led to publication of this report on human genome editing that discusses the science and the opportunities as well as the challenges of this technology especially in the context of human germline editing I think this is a great start but as we all are aware this field is continues to move very very quickly new technologies and and sort of improvements to the technology happening all the time and so there continues to be a need to discuss this and encourage more public contemplation of how we take appropriate stewardship of this technology going forward and and I just want to end by pointing out that as I said the field continues to move very quickly and one of the things that's happening is that as I mentioned at the beginning of the talk these CRISPR systems are very diverse in nature so it's possible to find new ones and this is taken from a publication of ours together with Jill Banfield from last year where we found several new examples of cast proteins that are RNA guided enzymes that have have no similarity to caste 9 but they function in a similar fashion so we know that there will be opportunities to find these types of enzymes and they may have new functions that allow new kinds of applications so it continues to be a really exciting field in terms of fundamental discovery as well as thinking about applications and how to how to make sure we have responsible use of these technologies so I'll just conclude by pointing out that RNA guided gene regulation occurs in a variety of biological context so this is kind of the you know the CRISPR caste system is kind of the latest example of RNA guided control of genomes you may have been a interference and eukaryotic cells so this is a clearly a very important way that cells can recognize nucleic acids and manipulate them in different ways we know that applications of genome editing will depend on both delivery and control and when I say control I mean both chemical and societal and then finally the fundamental research will continue to explore new CRISPR systems and what comes after CRISPR and so we just you know I think it's we're at a really exciting moment in biology when this technology coming together with others are putting tools in the hands of scientists that we've never had before and that really opened the door to doing all kinds of research and engineering and applications that would have been almost unthinkable just a few years back so with that I'd like to conclude by acknowledging a terrific group of people in my laboratory so I mentioned many of these students along the way and I also want to point out that we've had terrific collaborators as well beginning with Emmanuelle but also a number of people at Berkeley and I didn't mention Joel poleski but he's been working with us to develop Cass proteins for human papilloma virus detection it's something that we're hoping to be able to do eventually in point-of-care clinics finally we couldn't do anything without our funding agencies and this is where I really like to point out that I think you know the importance of supporting fundamental curiosity-driven science really can't be overemphasized it's a so critical that we have opportunities to work on problems that we think are interesting because as you can see in this story there's there's a certain serendipity to scientific discovery that really can't be predicted with that I'll close and I'd be happy to take questions if you have them thank you [Applause] well thank you very much Jennifer for that real tour de force that covered everything from fundamentals of bacterial phage infection and going all the way to structure and function to a wide range of applications and ethical issues so it really shows a tremendous breadth and you talked about serendipity but it does remind me of Louis Pasteur saying that chance favors the prepared mind and it was that ability to sort of see all those connections that's really very remarkable so it's now my before we go to questions I think I will do this first because I might forget later and so it's my pleasure to give you a scroll and a medal that goes [Applause] so floors now open for questions if people have questions could you you said you don't have the answer to the correct ethical way to use this very powerful technology but perhaps you have an opinion um what what is it well where do I start I think it's first of all critical that we work to develop a consensus among scientists about how to use this I think that many of us and certainly many people before me have concluded that it's very hard to imagine putting in place regulations that would be adopted globally and respected globally necessarily and how do how would you enforce such a thing so I think I think next best to that is is encouraging a scientific consensus about use of technology that provides a framework for how we deploy it going forward so that's really the goal I think of these kinds of meetings and reports right now is to encourage that and then I think the second piece is really to have an open discussion so that people who are not scientists are not blind sighted by what happens when technologies start to be applied in different ways so I think those are two of two important pieces to to how to proceed so I had a question you know from your fret studies and other biochemical data you showed it looked like even when there were mismatches there was some significant signal but the actual off-target sort of you know in fact is reportedly much smaller than I would have thought from that signal is that is that because the sort of homologous recombination is pretty inefficient and so it's sort of amplify is the real site compared to the I don't know what I think it's actually what I think it's due to is I think it's a function of using a minimal amount of caste 9 in cells so that you get you really minimize the number of off-target cleavage events and that's really been underscored I would say by a lot of the studies that have been done with enzymes that that are modified in different ways to try to make them more more accurate and what we often find when we test these biochemically is that they just they're just you just lower the overall level of activity so I think that you can achieve that in in a cellular setting by just reducing the concentration of cast line in the cells and so I think as people have gotten more sophisticated about how to deploy this in in those sorts of genome editing applications we're now working typically with limiting amounts of cast line so you really minimize off target events for that reason so it is the technology getting to the point where it's safe from an off-site you know off target point of view or how far away yeah I think the answer is is yes in the sense that what I've seen for data coming from primary cells so not not not cancer cells cultured in the lab but you know cells that are from primary tissues plant systems and and also human cells is that when you do deep sequencing looking for off targets using from cells edited using a minimal amount of cast line it's actually remarkably accurate so it's not that easy to find off targets and the other thing that that's done now that wasn't appreciated early on is that you can design guide RNAs to largely avoid most of the close matches in the genome and you know avoid off targeting for that reason so I think you know it's getting to a point where you can actually envision clinical uses it's not to say that we don't need to continue development but I think it's good enough now that people are really contemplating how you might employ it in a clinical setting hi Jennifer I wanted to ask you what's your opinion on ribonuclease targeting chime errors do you think that they offer a quicker route to therapeutics than the CRISPR technologies are you talking about RNA targeting versions of the cows proteins or no and ribonuclease targeting chimers ribonuclease sorry I'm targeting chimeras ribonuclease chimeras ribonuclease targeting chimeras ribonuclease targeting chimeras yeah so we've had two qui tam so I work for a company called frontline genomics and we've had some interesting articles about this new technology and maybe we could talk about it after day we will talk about it hello could you tell me what's your opinion about using CRISPR to modify people for fun like I don't recommend it I was wondering with the the taught you that there but the the template part of rnase 20 bases and with the start in motif I'd take it but it's always 20 bases is that enough to actually accurately edit the human genome I you've beef three billion base pairs you've already said that you can get off target because of the not matching completely but in terms of the template I take it is always fixed at twenty bases or can you change that for accuracy right so so it turns out twenty bases is just about right to get recognition of a single sequence in the human genome although again as I mentioned you can adjust the sequence of the guide RNA to ensure that you avoid targeting you know closely matched sequences and a genome like that but people are also fooling around with you know making longer guide RNAs and so far with cast nine that hasn't worked and the reason is that the protein protects 20 nucleotides of guide RNA and when we make extensions on the ends of the RNA they get nibbled off in a Cell probably by nucleases so it's sort of a architectural explanation for that but there may be natural systems that use longer guide RNAs we've been working with one recently in the lab that's using a 23 base pair guide so you know there may be natural systems that use longer RNAs and there may be applications where that's advantageous so it's any research to try and work out whether that's I plant a to sell people are just finding different you said there's different versions to cast nine different molecules are you looking at the accuracy of because obviously if you want to do editing you want it to be as accurate as possible of course yeah I think that's that's certainly a topic for a future investigation and currently what I can tell you is that you know it's one of these crazy serendipitous things but this enzyme that that Emmanuelle Charpentier and I started working on turns out to so far be still largely the best at this kind of gene editing and we think we have a biochemical explanation for that in work that I didn't talk to about to you about tonight but we think that this enzyme is a remarkably good helicase it's very good at melting DNA compared to a lot of the other natural variants of caste 9 for example found in bacteria and so it's not as trivial as one might think to just take new RNA guided enzymes and apply them for gene editing they don't always work right away and I think that has to do with you know at least in part with the biochemical behavior of these proteins thank you hi so I'm from the science policy Research Unit at the University of Sussex and I'm particularly interested in the security biosecurity aspect of CRISPR and how it relates to dual use research of concern do you think that there's a hype surrounding CRISPR being used as a biological weapon and how it can be used by non-acting stay non-actor not non-state actors yeah do I think there's hype yes I do because I think you know the you know no offense to any media that are here reporters but you know I think that those are the kinds of articles that that get attention but I also think that it's an important topic and something important to think about because there of course is potential for this technology to be used inappropriately and and unethically so this is one of the goals I think of the meetings that are ongoing is to discuss what those are and and try to mitigate it against it and again it's a it's a real challenge because it's you know the as you might have appreciated from this talk this is a technology that's widely available it's easy to get a hold of there now kits sold on the internet for high school kids if they want to you know make green fungi and things like that using CRISPR cast 9 so it's you know it's just I think it's a topic that is going to continue to require careful attention and the good news is there are lots of folks like you and your colleagues that are thinking about this and and discussing it and and trying to trying to figure out appropriate ways to control it hello there's been a lot of debate especially in that you are these products GMO not GMO cetera how should they be regulated what do you think is an appropriate framework and way to think about this technology and the products this technology and how they should be addressed I was fascinated to learn when I got into this that the way GMO is defined is different in different countries so in the u.s. GMO is defined according to the actual product that results from the manipulation whereas in in Europe the you know GMO is defined according to technology that's used to create something so you know the same product that in the United States would be considered non GMO because edits were made to the genome that didn't involve introduction of any foreign DNA for example would in the year in European countries be considered GMO so it's you know one of these kind of crazy things so I think that it really brings home a couple of things one is thinking about how we actually define something like that and it's a you know it's a bit of a technical thing it's hard to explain that to people that don't have a technical background with the differences but what I like to point out as well is that especially when we are thinking about GMO plants effectively everything we consume is GMO because it's been bred typically to have certain properties that we find desirable and plant breeders traditionally have used methods to introduce changes to DNA that involve either radiation or chemical mutagens that introduce all sorts of random changes to DNA they select plants that have desired properties but they don't control for what other changes in the DNA are coming along for the ride and so the I think the really powerful thing about CRISPR caste 9 is that this is now a technology that gives plant breeders a tool for making a precise single change to a genome or multiple changes but in desired places that don't bring along any other mutations in principle so I think this is really you know what people need to appreciate and this is what is I think under discussion is how we understand that technology explain it and then regulate it appropriately given the way it works there are a couple of questions over here hi you touched on the other nucleuses given that the vast amount of research goes into the standard Casa nine do you ever think from a commercial point of view that we'll see the other new classes enter the clinic oh absolutely I do yeah one over by the end there thank you for a wonderful particularly interested and you said that you actually said all these reactions don't external doesn't require an external energy source sternal yeah yeah so a lot of a lot of enzymes that manipulate DNA and especially those that that melt it apart use a external energy source so that means they use molecules of ATP or gtp to drive that kind of DNA unwinding and what's amazing about this protein is it doesn't do that so it has no need to hydrolyze ATP to drive this melting reaction it does it somehow through a series of conformational changes and probably taking advantage of the thermodynamics of RNA binding to DNA for the evolution of prokaryotic and eukaryotic cells if this doesn't require an external source I'd be hard-pressed to to sort of fit that into the sort of overall evolutionary argument for these organisms I think that it's possible that you know in bacteria that you know these these enzymes have to work fast they have to be they have to be very very fast and they have to be very efficient at DNA recognition and that perhaps precludes having to also bind to a second molecule of ATP to hydrolyze but I'm speculating for just two more questions one here thank you so much that was a really interesting talk I was hoping to live tweet if I was completely indulged experience my question actually follows on from my colleague from science Policy Research Unit so you discussed that there's been a meeting in which scientists and other people have talks about sort of the ethics surrounding the usage and you also touched on the fact that perhaps in the future we could design our babies oh wow so my question is in future discussions that you have surrounding perhaps governance or non governance what what sort of ethical codes or codes so what level do you talk about in terms of discussing the ethics of usage because you mentioned that it's not it be less about enforcing governance or regulation and more about an open discussion within scientists but do you think that needs to extend to perhaps state members who could benefit or use the technology for their own agenda so what what level of discussion do you think is that's in from your perspective in order to ensure that you know this this technology isn't used for but for bad things basically well I guess I I think about the I think about the example of in vitro fertilization so I'm old enough to remember before there was in vitro fertilization and then after right and so in my house you know growing up I remember my parents debating when the first you know test-tube babies were being born and my parents you know debating this and is this right and should people do this and you know and I think that was common in a lot of you know a lot of families and and groups and then what happened was that you know Louise Brown grew up and she was normal and other babies were born and they appeared to be normal and this gave an opportunity for couples that were infertile to have kids and there was such an incredible desire to do that that that industry really developed rather organically and I suspect that what we'll see with gene editing you know could take a similar path in the sense that you know there's already in vitro fertilization going on and people do pre-implantation genetic diagnosis to figure out if embryos have particular genetic traits and so we could imagine that going a step further we're at some point it becomes offered to parents in those sorts of clinics this opportunity to do gene editing and you know inside the question becomes you know how should we should we try to control that process or let it happen organically should we try to engage governments in this in this discussion in my experience I don't know what it's like here in the UK but in my experience in the u.s. I've been to Congress maybe three times and we only have one it was just start by saying we have only one PhD scientist in our entire Congress the shocking but you know in my experience so the the congressional members that I've met with are typically not sophisticated scientifically but they also are very very interested in this technology so they really want to understand it so I am sensing you know sort of a desire on the part of our government officials at least in the u.s. to figure out what's going on whether that will mean they actually put in place responsible legislation I wouldn't want to gamble on that one so I think you know again I keep coming back to the fact that I think scientists really need to be deeply engaged in this we need to be discussing it publicly we need to be sharing our information and we all just need to you know try to work together to figure out the best path forward it's a you know it's a complicated topic as you know and it's a really important one and it's one that you know many many people are very interested in which is okay so I realize this is a an endlessly fascinating topic that I'm afraid we have to bring the proceedings to a close and I want to finally conclude by thanking Jennifer Doudna again for really a splendid talk of recovering a huge range [Applause]

Channel: The Royal Society

Views: 33,931

Rating: 4.8949099 out of 5

Keywords: Royal Society, science, scientists, national academy, UK, crispr, gene editing, biology, jennifer doudna, genome, genome engineering

Id: oRz2vck3giU

Length: 79min 32sec (4772 seconds)

Published: Mon Jun 04 2018

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Gene editing with CRISPR technology is transforming biology. Understanding the underlying chemical mechanisms of RNA-guided DNA and RNA cleavage provides a foundation for both conceptual advances and technology development. Professor Jennifer Doudna ForMemRS discusses how bacterial CRISPR adaptive immune systems inspire creation of powerful genome engineering tools, enabling advances in both fundamental biology and applications in medicine. She will also discuss the ethical challenges of some of these applications.

The Croonian Medal Prize Lecture is the premier lecture in the biological sciences and is delivered annually at the Royal Society in London.