Professor Jennifer Doudna - CRISPR-Cas9: Genome Editing and the Future of Medicine

on this afternoon for this uh fantastically exciting lecture from uh jennifer didnow um i'm director of the welcome center for human genetics the center for personalized medicine is is a part of that out in in with anne's college and helen king together we chair the steering group of of the cpm and uh without further ado there's um an introduction from the stanley hall foundation who support this lectureship hello and welcome to the oxford center for personalized medicine lecture by professor jennifer daundell in honor of the memory of dr stanley ho i am ian hewan a trustee of the doctor stanley hall medical development foundation and ceo of the atorium group a nasdaq listed biotech company the foundation aims to enhance the quality of medical service and delivery in macau hong kong and other regions of china and enters into academic partnerships with relevant worldwide organizations to achieve this it is our pleasure to have supported the oxford center for personalized medicine since 2013. the university of oxford center for personalized medicine is based at st anne's college and the welcome center for human genetics uk its principal aim is to engage a wide range of interested people including academics clinicians students patient groups and the public with personalized medicine dr ho's support for the center marks his vision and commitment to help transfer knowledge of personalized medicine to health care professionals and students worldwide dr sennihoe passed away in hong kong at the age of 98 on may 26 2020 he was not only an extraordinary leader but also a great philanthropist his legacy is his extensive altruistic efforts from disaster relief to epidemic control having helped shape the communities and lives of many who have been touched by his generosity i have lost a lifelong mentor the foundation and i will honor dr ho's memory by continuing his spirit of serving our communities it gives me great pleasure to introduce tonight's speaker professor jennifer downer she's the lee kashing chancellor chair professor in the department of chemistry in the department of molecular and cell biology at the university of california berkeley professor downer has been a leading figure in the crispr revolution for her fundamental work and leadership in developing crispr mediated genome editing in 2012 professor downer and emmanuel chapentier were the first to propose the crispr cast 9 could be used for programmable editing of genomes which is now considered one of the most significant discoveries in the history of biology they award they were awarded the 2020 nobel prize in chemistry for the pioneering work professor download it is my pleasure to welcome you as the inaugural oxford cpm's dr stanley home memorial lecturer it's a great pleasure to be here virtually i am delightful and delighted to to be part of this extraordinary lectureship and to be the the inaugural lecturer it's really incredible legacy of dr stanley ho and i'm delighted to honor that i'm going to share the slides and um i wanted to start off by first of all just uh acknowledging oxford and thanking them for organizing this as a virtual lecture it was planned originally to be of course uh at oxford and we hope that will be possible again in the not too distant future but in the meantime i'm delighted to share with you some of the science that we've been doing in the laboratory and i thought i would uh break the talk into three parts i want to tell you first a bit about the backstory of crispr it's kind of an interesting example of the serendipity of small science really in discovering something completely unexpected in nature and then how it could be harnessed for a tech as a technology for genome editing that's what i'll talk about in the second part and then in the rest of the talk i want to tell you a bit about some of the research we're currently doing to understand how the crispr technology works and importantly how it can be deployed for clinical use and i'm excited to to say that there are already clinical trials underway using crispr and i'll say a bit about the the status of that work as well so uh you know crispr is a tool that allows genomes to be edited in a programmable way and i'll say a little bit about what that means what you're looking at here is a representation of the crispr cas9 protein in green with a molecule of rna in yellow that provides the zip code the molecular guide that allows this protein with its rna to interact with double-stranded dna and trigger double-stranded dna cutting and i have a video that illustrates exactly how this works so we have gas line with its guide rna engaged with a molecule of dna and if you watch you'll see the dna get cut and so this is a this is a system that is used in bacteria as a programmable adaptive immune system that allows bacteria to fight infection that's in fact how the technology was identified in the first place was by a small set of scientists who were studying this uh bacterial immune system to try to understand how it worked and so going back to the mid 2000s there was some interesting suggestive research primarily from bioinformaticians at the time including uh francisco mojica in spain who identified dna sequences in bacteria that were adapting in real time as bacteria became exposed to bacteriophage or viruses and this was a tip-off that there might be a mechanism by which these bacteria could store genetic information from viruses in their own genomes and then somehow use it to protect cells from future infection so this is a a video that illustrates how we now understand that crispr works and so you can see bacteria growing uh say in a biofilm and upon infection by a phage if the bacterium has a crispr sequence in the genome it can acquire a new sequence of viral dna and integrate it into this special place in the genome where each viral sequence is inserted between a dna a repetitive dna element the cell makes an rna copy of that that crispr sequence the rna is processed into shorter bits that each include a sequence derived from a virus and then together with a second rna called tracer and the protein cas9 this rna-guided surveillance complex is able to search the cell looking for a dna sequence that has a matching sequence to the rna guide when that match occurs the protein unwinds the dna and cuts it allowing the dna to be degraded in bacteria so this is a terrific way for these prokaryotic organisms to acquire immunity in real time as they become infected with phage so the biology uh here is very interesting and uh and then um as my collaborator emanuel charpentier and i were investigating the mechanism of this process it became clear that in uh that this double-stranded dna cutting activity of crispr castline could be harnessed in a different way in plant and animal and human cells because of the way that our cells deal with double-stranded dna breaks and that's shown here so in eukaryotic cells when dna double-stranded breaks occur the cell can recognize the break and repair it and in the process introduce a small disruption to a dna sequence or integrate a new section of dna that incorporates new genetic material at the site of the brain and this was research that you know had been going on for a couple of decades before crispr came along and many scientists had recognized that by introducing a double-stranded break in a in a genome for example in a human genome that it was possible to change that dna sequence during this process of repair however the challenge was how to introduce a targeted double-stranded dna break something that is obviously non-trivial there were earlier technologies for doing this that relied on programmable proteins like talons and zinc fingernucleases however these proteins took effort to engineer and had to be redesigned for every dna edit the amazing thing about crispr is that it's easily reprogrammed by simply changing the guide rna so here we're watching a video that illustrates the crispr cas9 protein with its guide entering the nucleus of a eukaryotic cell and then surveying the sequence to identify a sequence that matches the rna guide and as you can see this triggers dna unwinding locally it allows the two chemical active sites of the cas9 protein to cut the dna double helix precisely just like you would cut a rope and then those broken ends of dna are handed off to repair enzymes in the cell that can fix the break by introducing a small change in the sequence like in this example or by integrating a new piece of dna if there's a dna template available in the cell and so when emanuel sharpentier and i published this work in 2012 it was very quickly adopted by multiple labs around the world and now eight years later has actually gotten to a stage where in addition to being used widely for research there are multiple opportunities for clinical applications that i'll discuss uh briefly and so you know the the one thing to mention here is that we're focusing on applications in medicine but the exciting thing about crispr is that it's cross-cutting across essentially all areas of biology and so we over the last eight years there's been incredible advances in basic research as well as both medicine and agriculture that have relied on this fundamental ability to manipulate dna in a precise and positional way in cells and it's really opened the door to accessing the information in the human genome and other available genomes in a way that up until that point had been difficult uh or in some cases not really possible so in in clinical medicine i want to i wanted to say a little bit about um some of the incredible opportunities that are coming up for doing editing that will have an impact on patients and i think the vast majority of this type of editing involves somatic cell editing where cells from a patient can be corrected using a crisper-based approach and then used in those patients to correct their disease-causing mutation and this is a this is a an example in the case of sickle cell disease where the mutation that causes sickle cell disease has been well known for decades and it results from a single base fair change in the human beta globin gene that gives rise to a mutant form of hemoglobin that causes the the distinctive sickled shape of these cells and leads to a number of very devastating you know physiological effects on on patients including organ damage and great uh pain and very difficult disease to manage and uh it became clear really very early in the in the whole development of the crispr technology that this approach of crispr could be used to correct either correct this mutation directly or to activate a secondary gene such as fetal hemoglobin in these affected cells that would mitigate the effects of the disease and so that work which began in culture you know cells that were being cultured in the laboratory has now progressed to a point where it's being actually applied in patients and this is a great example of the first u.s patient to receive a crispr therapy for her sickle cell disease victoria gray who has made headlines over the last year and a half because she's done very well with this therapy and um and so there's been a lot of excitement about the potential for this treatment to be truly impactful in patients that are suffering from not only this disease but many other rare diseases that would otherwise typically be unaddressed and very difficult to either treat or in some cases even to to diagnose and crispr can can in principle do both and so one of the things that uh that i've been working on at the innovative genomics institute is how we think about a therapy like this from the standpoint of access because although the the the uh the therap the the technology is is exciting and we can see the potential to have an impact on people's lives i think that you know to make sure that this kind of therapy in the future is affordable to people that need it is is paramount and so that's one of the things that i'm doing currently is to really work on how we can make crispr into a technology that is accessible to the largest number of people and just for example this uh current therapy for crispr costs north of one million uh us dollars per patient so it's clearly uh at a price point right now that would not be accessible to people around the world that would potentially benefit from it another aspect of crispr that i want to briefly mention here is uh is the ethical use of a technology that in principle gives us the the power to rewrite uh you know the human uh genetic code in a way that could impact future generations and so one of the uh one of the the things that i've spent the last several years participating in are global conversations about the ethical use of crispr and in particular that this comes up when we think about applications in the human germline which means applying crispr in human embryos or eggs or sperm in a way that would create heritable changes in the human genome something that has been widely discussed and debated and there's a series of reports that have been released including the one shown here that was the result of an international committee led by the national academies in the u.s and the royal society in the uk so i think this is a very important topic to continue discussing and and to you know maintain transparency around its use because there's no question that this type of research certainly is going forward and i think in the future there may be opportunities to use crispr in this fashion but we have to make sure that that work proceeds with appropriate uh guidelines and responsibility um and so i want to just in the last part of the lecture i just i want to turn to what is currently going on in the research lab to address some of the ongoing challenges of crispr as well as how it can be used to address even the current coronavirus pandemic and um and i want to i want to really talk to you briefly about three different questions that we've been posing from a research perspective regarding the the accuracy and the efficiency of using crispr for any application but primarily thinking about it in terms of applications and clinical medicine and the first question is how do we ensure the precision of editing and so we are working on this in in a number of different ways but the uh short story i want to tell you today has been the result of a terrific collaboration with the lab of david liu at the broad institute at harvard that has involved investigating the the activities of engineered forms of crispr cas9 that are capable of chemically editing an individual or a set of nucleotides in dna that collectively are called base editors and just as an example this is a figure taken from a 2017 uh publication from the blue lab this shows a collection of pathogenic human single nucleotide polymorphisms and out of the total that were categorized here about half of them could be corrected if it were possible to convert an at base pair in dna to a gc pair and of course doing that site specifically is uh is a tall order and yet the the crispr technology is in principle capable of this kind of precision chemical modification uh if if it can be harnessed in the right way i'll show you very briefly the approach that the lu lab and the condo lab and others have taken to creating base editors from a crispr platform the idea was to take the crispr cas9 protein that i showed you before and make a connection a a protein linkage to a domain of an enzyme over here in red that is capable of of converting adenosines in dna to innocenes which can then be converted further to guanosines when the cell goes through a round of dna replication and dna repair and so this was kind of the you know the schematic of yeah wouldn't it be great if you know this this could happen and the idea here was that uh cas9 because of its mechanism that i as i showed you it's a protein that uses the guide rna to trigger dna unwinding that's catalyzed by the cas9 protein and then in the natural setting dna cutting and so in this instance what uh the the the idea was to remove the cutting ability of a cast iron or turn it into a knick ace so it could only cut one strand of the dna in a fashion that would tell the cell which strand of the dna was actually to be was the correct strand for dna repair and then allow this dna unwinding capability to expose a sequence of dna for editing at a particular position by this editor uh domain that was appended to cast 9. so that all sounds great uh the challenge was that there's actually or there were many challenges but you know one of them is that there's no natural enzyme in nature that has this chemical capability of modifying a single stranded dna to convert an adenosine to a to anything really but certainly not this pathway here however there is a domain there is an enzyme that's well known in bacteria that can do this chemistry in rna and this is a trna modifying protein called tad a that functions as a dimer its structure is well known and so the the strategy of the the lu and the condo labs was to use directed evolution in the laboratory to introduce mutations into this tad a protein and then select for enzymes that would have the ability to to do the kind of editing that i showed on the last slide and through a number of rounds of selection there were a series of these types of chimeric editing proteins that were appended to castline that were evolved in this kind of laboratory setting to do uh adenosine to um to innocent and then guanosine conversion in cells and so this was great uh from a sort of an editing you know technology perspective but the question was how to turn this into a ultimately into a tool that would be precise enough and and uh and effective enough that it would actually be useful in a clinical setting and so this is where we started to work with the lab and doing uh what we like to do which is diving into the molecular mechanisms of enzymes we did some time course measurements in the laboratory with purified versions of these different generations of editing proteins and we found that most of them were very very slow uh enzymes so this is looking at a time course here this is a one-hour incubation of a purify these purified editing proteins uh incubating with double-stranded dna and looking at the chemistry that's shown here that can be detected very nicely with a uh using an endonuclease cleavage assay and what was striking was that there was one editing protein that hadn't at the time been very well characterized from the lu lab called they called it a b e 8 e this stands for a base editor eighth uh eighth edition i guess and uh and this this uh protein had markedly different kinetics in vitro compared to all of these other a-base editors so we scratched our heads and said i wonder why that is and we start dug in to start figuring out what might be special about this particular version of the a base editor why was it so good at uh modifying an adenosine in this kind of a setting and so too one of the things that we wanted to do was to try to catch it in the act of actually editing dna so this led to a wonderful collaboration three-way collaboration with the lab of peter beale at uc davis who has a long history of working on that's had a protein from bacteria and they had done structural biology on this and had also come up with a very nice chemical strategy for trapping the tad a enzyme in the act of editing trna they could do this by substituting a nucleotide analog called eight aza nebularin which is shown here at the position of editing in a trna substrate and uh this would lead to a partial reaction in the presence of tad a that could not carry on to the the rest of the um uh you know to actually complete the chemical conversion of adenosine to to innocent and so the idea was to synthesize dna molecules that would contain as a nebular and at different positions in this part of the dna that we knew would become exposed as a single strand during cas9 binding and then see if we could actually capture the editing domain of this abe8e protein in the act of uh editing that position and so this was work done by a postdoc in my lab audrey lapine and cody palumbo a student with pete beale and uh after sort of uh summarizing uh two and a half years of work they eventually did find a position a modification in the dna that succeeded in in this kind of trapping and furthermore we were able to capture this and visualize it using cryo-electron microscopy together and this was work done by a second postdoc in the lab uh gavin knotts gavin and audrey worked together to do uh the structural biology on this protein and i'll show you just a a very short animation of this so you're looking at the cas9 protein in white the red and pink are the tad a editing protein bound to dna and hopefully you can see that the protein is holding on to its guide rna and magenta the dna is literally unwound as it traverses the protein and this is the important part this is really the the action area of this editor is the the loop of dna that enters the active site of the tad a enzyme and i won't show you in detail here but we could actually observe that the the uh the eight mutations that occur in the tad a version this eight a uh eight eighth edition uh version of the abe uh base editor are all clustered in the region of tad a that bind to dna and so it really was very clear that this enzyme has become adapted for a dna substrate it now precludes editing rna it doesn't doesn't edit rna very well anymore but it's terrific at editing this kind of looped out single stranded dna molecule and in fact it's almost too good because we did find and i won't show you these data but they're they're now published that this protein is actually also editing other positions along the dna probably as a result of the search mechanism of cas9 as it moves along dna looking for a target sequence to bind uh with its guide rna and so we think that there actually may be a happy medium to be still discovered that will identify a version of this modified editing domain that will be still fast and effective at editing dna but not so fast that it is able to to capture transiently melted dna during the cas9 search process so this is a illustration of how understanding the molecular mechanism of these types of proteins can in fact guide the direction and development of a technology like this i also want to point out that in nature there are a number of alternative proteins to the cast line protein that emanuel charpentier and i first investigated we've been interested in in finding these and understanding how they how they work and this has been a long time collaboration with a colleague of mine at berkeley jillian banfield who researches bacteria primarily growing in various environmental settings and she's one of the the international leaders in metagenomic sequencing which means collecting bacterial samples sequencing all of the dna in the samples and then reassembling genomes from those data to identify what organisms might have been growing in those settings and of course in the in the in the context of this type of work they also identify bacteriophage that are infecting these organisms and that was actually one of the she was one of the early labs to identify crispr sequences based on that type of work and so very recently um we've been working with her laboratory on a fascinating subset of crispr cast systems that are found only in phage so one of the you know surprises here is that an anti-phage mechanism of course has been picked up by phage and we think is being utilized by some phage at least to target their phage competitors and one of these proteins is a protein that we call caspi because this is a protein that is about half the size of the uh of the the original crispr cast protein it's about a 70 kilodalton protein and it's a very small crisper locus it has a just a single uh this one gene together with a crisper array of sequences and um it has a just a short guide rna that it utilizes for dna targeting and so in work that was done with bassin al-shayab a graduate student and patrick polish of postdoc we were able to start characterizing this crispr caspi system we found that this system is in fact the smallest so far that's been identified in terms of its total molecular weight compared to any of these other crispr cast type systems and yet remarkably it's still capable of uh not only targeted double-stranded dna cutting but also of inducing genome editing in human cells using a very simple assay that we can where we can identify and quickly sort cells that have been edited based on targeting of a green fluorescent protein encoding gene in these cells and this is just showing some quantification of different guide rnas that work in these cells and so we've been since this work was initiated we've been continuing to investigate how this very small protein is nonetheless capable of dna unwinding dna cutting and uh and triggering this kind of editing in eukaryotic cells and so recently patrick pousch and another postdoc in the lab kasia sulsek have been using cryo-electron microscopy to capture the caspe protein in in different structural states and i'll just show you one image here of the protein so we know that this we now know from this work that uh the protein is hugs the the nucleic acid very very tightly sort of wraps around the guide rna which is in orange in this image and the the targeted dna and we can see now in detail how the dna is recognized by this protein we have some important clues about how dna unwinding is triggered in these enzymes these very small proteins and we also have some data showing that these proteins are are naturally inhibited from cutting dna randomly by a structure that when removed makes these enzymes even more active than they are in nature so this is something we're currently exploring and i think is again illustrates the value of having this kind of mechanistic and structural data that we can use to understand the natural functions of proteins like this and then and then engineer them to do new things as needed for a technology development and then finally i wanted to briefly address this question how can crispr casts editing enzymes be delivered to specific cell types uh yeah that's i think this is the big uh current bottleneck honestly in the crispr field is is really figuring out how to do this i don't think there's likely to be a one-size-fits-all answer to it but um clearly having better ways to deliver these editors into tissues and cell types selectively is going to be key and just as a as an example going back to the to the the story of victoria gray and other patients who have been treated using crispr right now that treatment requires bone marrow transplantation and in those studies so far the the the primary health negative health impacts on those patients have been a result of the bone marrow transplant rather than apparently anything to do with the genome editing so it just speaks to the importance of figuring out safe ways to deliver these types of molecules in different settings and so what we're doing to address this right now is really thinking about ways that we can package the crispr protein with its guide rna for delivery and i wanted to just point out this paper from last year from pearl june's lab that was again you know one of the one of the highlights of last year in terms of developments in the crispr field because it really showed the potential to use crispr engineered t cells in patients who are suffering from cancer and highlights the the future potential of this kind of a strategy and as shown in this image this type of t-cell engineering today involves again x-vivo editing so the cells are removed from the patient the editing goes on in the lab and then the edited cells are returned to the patient and so we've been thinking about this and wondering if there might be an alternative in the future wouldn't it be great if you didn't have to do all of this and you had a way to provide a one a one and done injection or who knows maybe someday even a pill that could be taken that would um deliver the editors just to the cells whereas the team is is necessary and so this is a project being conducted by jenny hamilton and abby stahl two post docs in the lab and connor sushida a bio-engineering graduate student and the approach they are taking is to use virus-like particles as nice uh little packages for capturing assembled crispr cas9 proteins with guide rnas and then and the important thing here is that we can take advantage of the glycoproteins on the capsid surface for delivery into cells because this is effectively the viral infection mechanism importantly here these virus-like particles have been gutted of genes that would allow virus uh replication so they they're not infectious in that sense but they're able to transduce particular cell types and dump their contents into the cells and so the way this uh the way that we're doing this and and this has been a terrific collaboration with some additional scientists at ucsf in the lab of alex marson in particular david nyan and brian shy to md phds who are focused on um t-cell engineering for immunology applications and uh the strategy here is to use a it's kind of a one-two punch where we have cas9 protein rnase these are ribonucleoproteins or rnps that target genes important for this type of a t cell engineering approach together with a lentiviral genome that encodes a second a piece of dna that can target another gene in this case in a anti-cd19 which is cell surface receptor chimeric androgen receptor protein gene including this protein and so the idea here is to use the crispr cas9 system to target and knock out particular genes and at the same time allow integration of a new gene that can create that chimeric energy receptor so useful for for t cell therapies and so this is very briefly a summary of the strategy and so what we can do is to encode in addition to genes important for creating these lentiviral capsids we actually encode a chimeric gag protein that's been fused to cas9 and this is important because when these plasmids are transfected in the cells they create these kinds of particles in which the cas9 protein is anchored to the capsid so this ensures accurate and effective efficient encapsulation together with guide rna and then there's a proteolytic cleavage site that allows clipping of this assembled cast 9 off of the interior surface so that these are released inside the capsid and so we i'll show you a couple of um of data points here and so that we're able to use these this type of a strategy to get pretty robust uh expression of this chimeric androgen receptor in these targeted cells and um and we can quantify this in different ways so this is showing uh a quantification of two different uh types of t cells these cd4 positive cells and c8 positive cells uh shown here and importantly we can also engineer the capsid of these virus-like particles to target just one type of t cell and so this is an experiment in which it was possible to selectively target just the cd4 positive cells in a mixture of cells that also contain cd8-positive cells and so we think this is a strategy that in the future is has real potential to allow a targeted editing of just those cells that will be clinically beneficial in a patient while leaving all the bystanders standard cells unmodified and then finally just one i just wanted to mention also that we have a lot of interest in applying this this strategy of protein rna delivery in for genome editing in the brain and that sounds like a like a high bar and it certainly is but i think there's such an important unmet medical need right now for treating patients that have neurodegenerative diseases that were we've been very interested in this and this is actually a project that started several years ago in the lab with the work of a former postdoc brett stahl who came in and was able to show that he could engineer cas9 with peptides that were appended to the protein surface that allow cell penetration so this was a kind of a direct protein delivery strategy where these engineered cast line and guide rnas are injected across the blood-brain barrier this is an experiment conducted in a mouse uh using a reporter mouse where editing leads to cells that are red and so it's very easy to see cells that become edited and you can see a nice kind of dose response here that the higher the amount of injected engineered cas9 the more editing is observed and since then we've been able to improve the steadily improve the efficiency of this and uh and achieve really quite uh decent levels of editing again in the mouse um we're still investigating how this would work in a larger brain like a larger sort of animal brain but but i think the principle here is that we think that this will be a useful strategy for especially for targeted delivery where there's a desire to edit cells in just one region of the brain for example the kind of therapy that might be useful for treating something like parkinson's disease um and then i'm just one final slide i wanted to mention a crispr and coronavirus it's a little bit hard to uh you know sort of imagine uh the the the the uh complexities of the the past year for for everybody really around the world but um when the pandemic was just getting started in the u.s just about a year ago we realized at the innovative genomics institute that there was important some really important things that we could do as an institute to address this emergency and one of the things we did was to set up a clinical testing lab just using a standard pcr-based technology but that's been incredibly important because we're now a clinically approved laboratory that is able to test all of the samples from people across our university campus as well as we work with a number of healthcare partners in northern california that has allowed us to provide regular testing for people that would otherwise not have access to it for example folks that are that are unsheltered folks that are first responders of various kinds we've worked with a number of our fire departments and police departments to provide them with regular testing and and in the process of doing that we also realized that crispr could have an important role to play in testing and diagnostics as well based on some interesting chemistry that emerged from the lab from our lab several years ago now starting with a graduate student alexandra e saletsky who first showed that you could use the activities of some proteins in particular a type of protein called crispr cast 13 that is naturally an rna targeting form of crispr to report on the presence of the sequence and that very briefly the way this chemistry works is that these what alexandra isletsky showed is that these proteins naturally have an ability to recognize rna with an rna guided mechanism so very analogous to what i showed you for castline except that in this case recognition of a sequence turns on a non-specific rna cutting activity that will cut any single-stranded rna in the vicinity of this enzyme and so she realized this was also published at a similar time by uh gutenberg and feng jong but what alexandra recognized was that she could use this activity to report on the presence of a sequence if she had a quenched fluorophore pair of dyes that were coupled to single-stranded rna and so this was a this is a reaction then upon activation by recognizing a target this enzyme starts cutting the reporter and releasing fluorescent signal that can be easily detected and this fundamental strategy has now become widely adopted by a number of labs and companies there are second a second class of enzymes called crispr cats 12 that have a similar capability except for dna recognition so they have a dna cutting activity that gets turned on upon binding to dna sequences and so this is a great way to use crispr as a diagnostic tool and so we've been we've been uh recently this is a very recent publication that um is from the lab of melanie ott at the gladstone one of our long time another one of our long time collaborators to use crispr as a tool for detecting viruses and you may know the coronavirus is an rna virus so having a technology that can directly detect rna without having to go through a a dna you know step where you have to copy the rna into dna has some real advantages in terms of quantification as well as speed of the test and so that work is very much very actively uh under development and in fact we're excited that at our clinical testing lab we're just about to start using a crispr based uh laboratory test for uh coronavirus that will begin one of the beta test sites for a local company that's been developing the technology and we'll see how it works with our robotic pipeline in the clinical lab um yeah so i just want to wrap up there and just point out that you know i think continued mechanistic dissection of these systems will guide the further development of fast and accurate genome editing proteins we think that continued discovery of new genome editors including those that are very small and compact will give us new strategies clinically especially for delivery and finally that figuring out better ways to deliver these proteins especially in situ will be fundamental to the to the you know the continued advance of this as a as a real clinical therapy for lots of other diseases in the future i want to thank a great team this is obviously a pre-pandemic a photograph including all of my i think i had 15 undergraduate students that were working in the lab that summer so this is uh this is a showing the entire team here and i just want to give a shout out to our uh our original work with emmanuel charpentier with martin yannick and chris chelinski who did the work on cast nine originally our labs at berkeley and gladstone that are doing the the research that i talked about today from from our group as well as our collaborators alex marson melaniatt and also bruce conklin at the gladstone institutes and of course all of this is made possible by support from these agencies here thank you very much and of course i'll be delighted to address questions that you might have thank you jennifer wow so humbling and inspiring all at once illustrating fantastically the combination of biochemistry and structural biology coming through to that understanding to lead to the advance we've got um we've got quite a few questions and we've got a good 10 minutes um so many of them that you came in early you've asked you've already answered in your presentation about how merit help in cancer um there's one of the common questions what what would be one of your what would be your main concern of any going forward for the future of of using crispr technology um well i guess i have i have two one is one is that i i think that you know as exciting as it seemed to as it's been to see the the rapid advances i think there's also a you know needs to be appropriate caution with a new technology so um you know we want to make sure that as these especially clinical trials advance that that there's you know there's appropriate sort of restraint i guess to make sure that we don't get ahead of the technology i think so far that's been true and i've been pleased and i i hope that that continues and the other thing i'll mention briefly that i you know i do i do think about and i i feel uh some uh angst about is the accessibility as i mentioned i think that you know for this technology to really become impactful uh for people who need it it will need to really come down and cost and it needs to become much more accessible and that that's going to require not only delivery approaches but also ensuring that of course it's accurate and safe and effective and um you know that that that it can be tested safely in you know for many different applications which is another challenge i didn't talk about it but if you think about it also is an interesting challenge for a technology like this yeah one of the questions was how do you direct um the therapeutic to specific cells and again you address that uh there's a lot of work to be done there but um it looks if it started and and will be really important to get cell specificity on delivery yeah absolutely yeah um and and also i i think the approach taken again illustrates how sophisticated and how much thought goes into engineering to get that specificity that was really impressive um what one of the i know microbiome sequencing has taken off bgi the million human microbiome project but of course what's waiting in the wings is is the characterization of probably of millions of more phage uh so it it again you you mentioned this it seems almost unlimited functionality in and all the phages we've yet to discover it's kind of mind-blowing isn't it i mean it is mind-blowing yeah i hear this regularly from my my colleague jillian banfield who does this work you know she's always telling me jennifer we're just at the very very tip of the iceberg and the iceberg is huge so yeah and um there was one question about is is there any possibility of anti-drug antibody responses so they the host immune system reacts against one of the proteins that's been brought in yeah that's a very important question and and and uh and abs and the answers is absolutely yes and there are several publications already that you know show the potential for that for that um of course you know we're using a foreign protein here right so you can imagine that could be an immunogenic and not only that but you know the the most commonly used form of crispr the crispr cas9 protein is from it comes from a pathogenic bacterium that many of us already have antibodies to and so you could imagine that that could also be problematic in you know in people it's not been demonstrated to be the case but it's something that you know i think requires careful careful uh attention um we have a collaboration with a couple a couple of immunology labs at berkeley who have been looking at this question and so they have in working in a mouse model they have found that uh yes indeed that not surprisingly that these bacterial proteins are immunogenic but also that they can identify the immunogenic epitopes on the surface of these proteins and um they've been starting to engineer them to be uh you know be sort of humanized in ways that people have done effectively for antibiotics so i think that you know there may be opportunities there and then also i didn't mention it but the other one of our other motivations for studying these alternative crispr cast systems is that if they come from uh phage that you know have never uh you know infect a bacteria that have nothing to do with human physiology then we think that they may actually be intrinsically safer because they won't there won't be pre-existing antibodies in people yeah and the the same group published a very nice science paper recently that the same group that developed in germany developed the the visor messenger and they they the title of the paper is a non non-inflammatory messenger rna so not to a self antigen so in parallel there'll be better ways of tolerizing you to the key proteins that that you guys and others develop my my colleague peter donnelly who was the former director of the welcome center here in oxford and also co-chair of of the cpm he's he's asked a question the human genome is a big one how how does how does the cast 9 system find its sequence that is such a great question i would love to know the answer to that um yeah i mean that's you know um i actually just just hired a new postdoc from the lab one of the you know really top um imaging laboratories who does single molecule imaging we're hoping to start to dissect this by doing some live cell imaging but right now there are a handful of publications about this and you know there's been some work we did some work a few years ago with the lab of bob teigen on this topic where it's possible to put a fluorescent label on cast nine and then kind of watch its you know movements around uh cell nuclei but those experiments so far have just uh kind of told us the outer boundaries of you know how this how this works we don't know yet in detail uh how how it actually finds a target and one of the here's i'll just leave you with an interesting conundrum and that is that uh you know there there are a lot of uh potential uh sites of targeting for cast nine in a cell and if it's spent too long on any one of those you could imagine it would literally never ever have time to get to its true target so this has to be a very very fast protein and understanding how that works i think is still really a fundamental aspect of the field wow and and and it looks as if with the latest technology you can image it as it travels across the genome yes yes so i think there's a lot of opportunity there now with the breakthroughs and imaging technology for sure yeah um so um maybe going back to the very beginning um somebody's asked of of the several cass enzymes 12 6 13 9 how did how did you settle on nine yeah god that's it yeah that's another good one um well i have to i have to uh give a shout out to emmanuel uh charpentier here because um that that work really initiated when she and i met at a conference and she had been already investigating uh this crispr system in streptococcus pyogenes uh in an infectious uh bacterium that infects humans and um trying to figure out how its crispr system was working and they it used a cas9 based system and at that time my lab had been doing work on other types of crispr proteins to knock out and she said you know i think i think cas9 is a you know it's a very it's clearly very important uh genetically in these cells but we don't know what it does biochemically so we want to work together to figure it out and uh you know that's how we got into it but it was clear from already in the field at the time that there that there was something very very interesting about a single protein cas9 that had this art somehow this rna targeting capability where it could you know recognize a dna sequence and cut it it's just that nobody understood the mechanism and that's where we got involved in it yeah and and related to that i guess i mean is there a cast 24 when you characterize presumably there's millions of bacteria that remain to be found and correct sequenced yeah well uh there's there's definitely a lot of diversity of crispr systems for sure i think what's emerged so far in the field is that um the cast 12 family of proteins that i mentioned it just varied briefly but that turns out to be uh quite a large super family of different flavors of those casserole proteins so that's been kind of expanding over time uh whether there'll be a yeah is there going to be a you know a cast 15 16 17 18 24 58 i don't know my guess right now is probably not but if they're if they are there they'll be rare i think you know but again i gotta remember that you know we're at the tip of this you know huge iceberg of of uh of bacteria so what else is out there yeah we don't know yet and of course another impressive feature was that that evolution in the laboratory searching for variants that do what you want of the existing cast genes and that that that that also was incredible so we're um we've got through the questions um we've got i'll ask you one more like um when you must give this seminar a lot what what what's the toughest question you're asked well i think i think the i i guess i would say two tough ones are um are about ethics so nobody asked in this uh seminar about you know editing human embryos but you know that's one that's been on many people's mind you know do we have to worry about rogue use of crispr cass and my answer there is uh yes but i but i think that uh you know the potential of the technology is so huge and it's so important that we don't want to slow it down we just want to make sure that we um you know i think the global scientific community needs to be making a strong stand about you know how we want to see the technology proceed and i think that's been happening so i i'm you know when there's never a i don't think there's ever a way to enforce global regulations we have hard time even doing that in in particular countries but um but i do think that having um some clear criteria that are agreed to by authorities worldwide is really helpful and that's what's been happening with that um the other the other uh tough question i guess is about patents and intellectual property so nobody asked about that either yeah there is a question on that yeah yeah and and uh you know and i think i think the the challenge there is that um you know this is a technology that i think all of us i think would want to see adopted as widely as possible right and you know and and and developed as quickly as possible to be beneficial to the largest number of people and uh and the question is always how to how to do that you know appropriately and and to allow investors who might be developing commercial ventures to recover their investments which is really the purpose of the patenting system and so um you know i've i've really been pleased that the science i think of crispr has continued to develop quite you know quite quickly independent of the ongoing patent disputes which unfortunately i think are gonna we're gonna those are gonna be with us for a while you know they're gonna go on and on and on and lawyers are gonna are gonna be the winners there for sure um but you know but i think i think fortunately the science and and the application of it continues a pace and so um this this is really key to keep in mind you know when you read about the latest in the patent fights in the media remember that you know labs worldwide are continuing to do their work and this is not slowing any of it all right we've reached six o'clock uk time um thank you very much indeed fantastic thanks

Channel: CPM Oxford

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Length: 60min 42sec (3642 seconds)

Published: Tue Mar 02 2021