Distinguished Speaker Series - Jennifer Doudna, Ph D

[Music] good morning and welcome i'm allison beshear the dean of the uc davis school of medicine and we are excited to launch the uc davis office of research and school of medicine distinguished speaker series in research and innovation i am a strong believer in the importance of academic medical institutions and multi-disciplinary research in discovering life-saving treatments and groundbreaking healthcare innovations the pandemic has made it even more important to translate our leading edge research to the classroom and to our patients bedsides so that we're giving all of our patients access to the safest most effective treatments and care as one of the country's leading medical schools ranking seventh in training of primary care physicians the heart of our mission is to provide our patients with the best patient-centered care possible research innovations are a key ingredient to making that happen now it is my pleasure to introduce uc davis vice chancellor for research prasant mahaprabhu thank you dean brescia and welcome everyone it gives me great pleasure to introduce our guest guest speaker for today's distinguished speaker series in research and innovation at uc davis dr jennifer doudna is the lee casing chancellor's chair and a professor in the department of chemistry and of molecular and cell biology at the university of california berkeley she is also an investigator of the howard hughes medical institute her co-discovery of crispr cast 9 genetic engineering technology with collaborator french scientist emmanuel sharpened here has changed human and agricultural genomics research forever this genome editing technology enables scientists to change or remove genes quickly with the precision only dreamed up just a few years ago labs worldwide have redirected the course of their research programs to incorporate this new tool creating a crisper revolution with huge implications across biology and medicine just recently dr doudna along with her colleague received the nobel prize in chemistry for the year 2020. in addition to her scientific achievements and eminence she is also a leader in public discussion of the ethical and other implications of genome editing for human biologies and societies and advocates for thoughtful approaches to the development of policies around the use of crispr cas9 jennifer has received many prizes for her discoveries including the japan prize kavli prize wolf prize in medicine and i'm sure i'm missing out a long list of prizes in 2025 sorry in 2015 c was named by the time magazine as one of the hundred most influential people in the world dr doudna we are honored to have you here with us today we have more than 2 300 registered attendees who are very interested in what you will share with us the floor is all yours thank you preston for that very generous introduction and i want to give a special thanks to dr ralph green for the invitation to come and speak with all of you it's a real delight to be here and i can't tell you how excited i've been about the partnership between uc berkeley and uc davis especially over the last few months it's been just extraordinary and i'll say a bit more about that today so let me share my screen and i'm hoping everything looks good so what i want to talk about today is is crispr and coronavirus and and i'm going to break the talk into three parts i want to start off by talking about the fundamental science behind crispr genome editing because to me this really illustrates a classic example of why curiosity driven research is so important and for many of you who are watching who are starting your your educational course and your your your academic careers and thinking about what you want to do in the future i hope to convey to you how exciting it is to do discovery research because you don't know what is out there and the reason that we do this kind of work is to answer our fundamental curiosity about the world that we live in and in particular our own place in that world as biological beings so i'm going to talk about that the origins of crispr i want to say a little bit about crispr and genome editing and where that is going and it's a you know very fast-moving field with lots of exciting opportunities and applications and then in the third and and longest part of the talk i actually want to turn to where we are today and facing the pandemic and some work that's going on right now to apply crispr as a molecular diagnostic and i think this really illustrates the both the power of fundamental science but also the importance of collaboration and partnership in doing science that brings fundamental discoveries to bear on real world problems and applications so where did crispr come from so for those of you that have heard of the the acronym christopher you may or may not know that it actually describes a bacterial immune system a way that bacteria are able to fight viral infection and this is a scanning electron micrograph that shows the surface of a bacterial cell that's being infected by phage and these uh this infection process much like a viral infection of any type of cell or organism involves a battle between the host and the virus and so in the case of microbes these cells are growing very quickly they have very little time to fight off the virus and so over the course of evolution bacteria have evolved uh a number of different mechanisms of defending themselves against viruses and a number of these have been well characterized in in uh you know in molecular genetics over the past few decades but there was a system of adaptive immunity that had escaped the attention of scientists until around the mid you know early to mid 2000s and this was a system that um that is illustrated here in this cartoon in which bacteria are able to adapt in real time and acquire immunity to viruses as they infect these cells and and very briefly the way this works is that cells possessing a crispr immune system have a dna sequence in the genome that is quite distinctive and in fact this was one of the first tip-offs to bioinformatics researchers that bacteria might have a system like this because they noticed a pattern of repeat dna sequences shown by these r's that flanked inserted spacer sequences in the genome and these short spacer sequences turn out to be derived from bacteriophage the viruses that infect these types of cells and so this was uh this was very interesting there were three papers published back in 2005 that reported this observation they were not in fancy journals they were published by groups in working in the dairy industry and in the food industry who are trying to understand how this bacterial defense mechanism might work and what emerged over the next few years is that cells with this type of sequence in the genome are able to use it as a mechanism for defense against viruses and the way it works is that these cells make an rna copy of the crispr array so a transcript shown here they process that rna into short units that each include a sequence derived from a virus shown by the colored bars and those rna molecules assemble with one or more crispr associated or cast proteins to form an rna guided surveillance complex they can search the cell looking for matching sequences typically double-stranded dna and when a match is found allow the crispr cast protein to cleave the the matching dna sequence and this is a video that just illustrates how we imagine these systems working so here are some bacteria growing in a biofilm let's say and upon infection by a virus if the cell has a crispr sequence in the genome it can integrate a sequence derived from the virus into the crispr array and this integration process maintains the pattern of repeat sequences that flank these inserted viral sequences so that marks the locus as a special place in the genome from which a crispr rna transcript is generated and then processed into these unit length rnas and in some systems these rnas these crispr rnas combine with the second type of rna called tracer and a protein called cas9 to form the rna-guided surveillance mechanism for this immune system and these proteins then search the cell looking for matching sequences of dna when they find a complementary sequence there's a a cleavage reaction that's triggered in the matching dna and in bacteria those double-stranded dna cuts trigger rapid destruction of the viral dna so it's a really nifty way that cells can adapt and become immune to bacteria in real time and then pass that protective sequence information on to their progeny so in the course of studying this this immune system um with my colleague emmanuel charpentier we realized that in in in eukaryotic cells in animal and plant cells human cells double-stranded dna breaks are handled very differently than in bacteria namely they're recognized and repaired by mechanisms illustrated here either through non-homologous end joining that leads to a typically a small sequence disruption in the dna at the site of the break and also to homology directed repair if a donor dna molecule is present in the cell and can be integrated at the site of the double-stranded break and this was work done by many labs over the previous two or three decades to understand this type these different pathways for dna repair and eukaryotic cells and along the way scientists had recognized that you could harness this these repair mechanisms for genome engineering if you could trigger a double-stranded dna break at a desired place in the genome and that's where crispr cast nine comes in so in the research that we did uh together with emmanuel sharpenta's lab and our uh our lab members martin yaneck and chris tylinski they figured out that crispr cast 9 is a dual rna guided protein it uses the crispr rna molecule here for interaction with a 20 base pair sequence in double stranded dna and importantly this protein also requires the tracer rna which is important for stable assembly of this surveillance complex and martin yinnick in my lab a wonderful biochemist and structural biologist realized that he could link together the two natural rnas in the system to form a single guide transcript that had the targeting information on one end and the structural handle for cast nine assembly on the other end and for us when martin did these experiments with the single guide rna and showed that we had understood the mechanism of cas9 programming with rna and that we could control its cleavage at different sites in dna by simply changing the sequence in the single guide rna this was for us that kind of proverbial moment when a curiosity driven project morphed into a project that we realized was going in a very different direction because of the exciting potential for this to be a powerful tool for manipulating dna sequences with um with a precision and a simplicity that had never before been possible and so this was really the you know kind of our uh transition from you know studying these systems purely for um the purpose of understanding fundamental biology and bacteria to recognizing that we were on to a very exciting powerful technology for genome editing and just to illustrate briefly again how we imagine this works in eukaryotic cells that um of course in in eukaryotes the dna is in the nucleus and it's packaged as chromatin and amazingly the crispr cas9 enzyme with its guide rna is able to enter the nucleus with a nuclear localization signal that's been engineered onto it and search the genome for a matching dna sequence matching the guide rna when that match occurs we know the dna melts in the inside the protein the protein is able to cleave the the dna at a precise position making a blunt double-stranded cut and then the ends of the dna are handed off to repair machinery in these cells to fix the break by introducing a small change as in this example or by integrating a new sequence of dna if homology directed repair is implemented and so this has become a powerful way to manipulate genomes of basically all types of cells plants animals and and of course human cells with lots of exciting opportunities and implications for research but also for um very practical kinds of applications of genome editing that i'll say a little bit about um and in particular you know a lot of attention has focused on how one might imagine using this kind of technology to correct disease-causing mutations at the source and one of the one of those types of very well characterized genetic diseases that has been known for a long time is somatics is a sickle cell disease which is a great example of how somatic cell genome editing could be impactful in clinical medicine and you know this is a just a cartoon that illustrates the the sickle cell mutation so this is a single mutation in the human beta globin gene that gives rise to mutant form of hemoglobin that is prone to aggregation and so in patients that are affected with this disease they suffer from uh cells that have this classic sickle shape and they go through crises in their disease in which there's uh there they are subject to severe pain and certainly to um to tissue damage and with with the crispr cast 9 we now have a technology that in principle offers the potential to cure this type of mutation and disease at its source by making a correction to the genome either by directly altering the sequence of the affected the affected gene or by activating the expression of fetal hemoglobin which is also an alter a strategy for dealing with sickle cell disease very exciting this is now not no longer just an idea or a proof of principle experiment in the laboratory with cultured cells but in fact has gone into clinical trials and over the last year we've seen multiple announcements including one for a patient named victoria gray whose sickle cell disease has been effectively cured using crispr and you know very exciting sort of advanced for the field and i think many of us feel the uh the you know the encouragement and the potential for this to have a real impact on many people who are affected by genetic disease and even uh even rare disease for which this kind of technology in principle offers a personalized approach to treating or curing their disease i think about this more and more in the context of affordable access because as exciting as the announcement about victoria gray was it was also clear that in the current implementation of crispr for genetic disease it's simply too expensive for most people to to benefit from it and so i think a lot about this in the context of you know as a scientist what can we be doing to make sure that we are working to make this technology available to those that need it and i think uh one of the keys to this from a scientific and technical perspective is delivery delivering the genome editing molecules efficiently and effectively into patients we need to be able to do cell type specific editing we need to be able to make targeted genome edits in the cell populations and tissues where those edits can be beneficial and there's a lot of innovation that that is still needed in this field so again for all of you that are starting out in science and and thinking about what you might want to do in the future this is this is certainly one area where we we need you i'm i'm uh i'm excited to be working on this problem with colleagues at the innovative genomics institute where we have a very active program on cellular delivery with the goal of of creating strategies that will avoid the need for bone marrow transplantation for patients that are affected with diseases like sickle cell and will avoid the the expense of course of that uh type of of uh procedure but also the uh the um you know the health challenges to patients and going through that kind of a procedure so that's something that i work on very actively currently um and um and the other aspect of genome meditating for for clinical use that i've been working on quite a lot over the last few years is thinking about safety efficacy and and of course responsible use of genome editing because with a technology like this we have both extraordinary opportunities but also real risk in in terms of safety and and i would say ethical use of genome editing and this has come up in particular with the possibility of using genome editing in human embryos and so that's something that i think needs very active discussion and attention and i've been working on this with um our partners internationally and and and working towards creating a transparency mechanism so that folks that are working on research on human embryos with gene editing have a a way to communicate about their work and and make their work publicly accessible so i wanted to turn now to uh talking about crispr and coronavirus and um and by the way i'll just just interject one other thing and that is that um i didn't mention it here and i didn't put slides into the talk about this but i personally am also very excited about the uses of genome editing in agriculture and we have a couple of active programs right now through the innovative genomics institute that involve folks at uc davis partners pamela ronald and others who are working on on genome editing and rice and for other applications that i think will be very impactful globally in dealing with climate change and some of the challenges that are arising very quick quickly with that so i won't say more about that here but i do want you to know that that's a that's a very exciting area that i think is also one that will be impacted by the crispr genome editing technology now with the coronavirus so i want to point out that crispr cast systems in bacteria are widely diverse and this is something that's been quite interesting just from a fundamental science perspective a lot of the work on this that i have access to is through the research of jill banfield at berkeley a very talented scientist who has been working for a long time on metagenomic sequencing of bacteria particularly environmental bacteria and um and then also of course with our collaboration with emanuel sharpentier on crispr cas9 and so we find that these systems are diverse they have diverse functions they have diverse biochemistry all of them use rna guided recognition so they're all kind of you know sharing that core molecular principle of recognition and interestingly as we have discovered recently both bacteria and phage can encode these crispr systems so interestingly bacteriophage although they are the targets typically of crispr casts they can carry around these systems in their own genomes and use them to target other phage or even potentially to control gene expression from their hosts so i think still a lot of really interesting biology to be dissected there and so over the last roughly decade or so in my lab at berkeley we have continued to investigate this fundamental biology and as i'll show you now every now and then the you know those experiments reveal things that are unexpected and un or unpredicted about the behaviors of the of the proteins and the and the rna molecules that are part of these pathways that in turn have implications for technology and one example of this is research that was done by a former graduate student of mine alexandra east celeski when she was in the lab a few years ago and so she was studying a christopher cass protein called cast 13 that had been reported to be an rna targeting uh version of a crispr cast enzyme by this group here and so this is how these proteins work so they're encoded in genes adjacent to a crispr array in the bacterial genome and then these these proteins these cast 13s are expressed they bind to the transcripts made from the crispr locus and they have an enzymatic activity that can produce the mature crispr rnas that form these rna guided surveillance complexes so that was sort of interesting and then in work that was done by alexandra she figured out that these proteins have a second enzymatic activity that allows them to cut non-specific rna molecules upon activation by binding to a target sequence so target recognition triggers cleavage of single-stranded rnas that are found in the in in the immediate environment of the enzyme and you can imagine that in bacteria this might be quite useful during an infection where recognizing a transcript from or even a section of a viral genome will trigger rapid degradation of lots of rnas that are in the in the mix and and triggering um either cell attenuation or even cell death if if the if the uh cleavage activity is significant in the in these cells and so um as alexander was doing these experiments we were you know kind of intrigued at this this kind of non-specific rnase activation and we started talking about you know i wonder wonder you know why why do why why do these proteins do this and also would is that useful at all and it was really in a a conversation with my spouse jamie kate who is also a professor at berkeley who said i think this would be a really interesting way to have these enzymes report on the detection of a sequence by designing a system where the activated ribonuclease activity the non-specific activity would cleave a molecule an rna molecule that was labeled with a quenched fluorophore pair where upon cleavage the fluorescent signal would be released for detection and so uh we literally you know did the simplest version of this experiment that you can imagine initially which was to go to the freezer and pull out a reagent called rnase alert that some of you might have used in the lab if you are trying to see if your buffers are contaminated with ribonuclease and you can and then these these assays are are set up with single-stranded rnas with quenched fluorophore pairs where rna's cutting releases a fluorescent signal and you can see that if you sure enough if you add ribonuclease a to rnase alert you get a very nice fluorescent signal and so we did the same reaction except using various crispr rnas that were were programming the cast 13 enzyme to recognize different viral targets and these are all bacteriophage lambda sequences and testing various both on target and off target sequences and what we found is that we got very nice triggered very nice signal detection using this rnase alert assay down to about picomolar levels of detection and so that was that was kind of cool and you know sort of one of those experiments that uh you know works the first time and made us realize okay i think we really do understand how these these uh proteins are working and um and then alexandra graduated and um um and another student came to the lab janice chen and she uh stumbled across another surprisingly sort of parallel type of activity in a different class of crispr proteins called cast 12. and these are these proteins are similar to cas9 in the sense that they use an rna guided mechanism to recognize a double-stranded dna target and make a double-stranded dna cut but in experiment an example of which is shown here janus chen found that these proteins also have the ability to cleave in this case a single stranded dna substrate non-specifically after being activated by recognition of a target sequence and this is just using an m13 phage which is a single stranded dna phage and what you can see here is that with cast 12 upon recognition of a target like this the enzyme is able to cleave single stranded phage very efficiently and the dna is essentially shredded this is an activity that is completely absent from crispr cas9 we don't see that kind of cleavage of single-stranded dna at all in these in these systems and and then this is just showing that this activity is indeed a target activated single-stranded dna activity so here's another experiment that janice chen did where she allowed the crispr cast 12 enzyme to recognize a double-stranded dna target this is a plasmid molecule and you can see that in an agarose gel we get cleavage of the plasmin so we get the kind of expected double-stranded cutting that we would predict for this enzyme and then if you add a radio-labeled substrate to the reaction you can detect very rapid cutting of non-specific single-stranded dnas after activation of the enzyme but no cleavage of non-specific double-stranded dna so this is still a double-stranded you know targeted type of cleavage mechanism it's just that the activity is able to also cut single-stranded dna that might be uh found in the in the uh environment uh you know so the nearby with the uh and able to access the enzyme active site why would why would cells want why would why would crispr systems be set up like this well we speculate that this kind of activity again provides cells with an additional nuclease activity that can shred single-stranded dna that might uniquely correspond to a viral single-stranded phage genome and not harm the the host chromosome because it doesn't impact non-specific double-stranded dna so again janus thinking about you know sort of how this kind of activity might be useful as a detection mechanism designed an experiment to actually try to distinguish between the viral speech strains that infect humans in in the case of human papilloma virus and so this is a this is a sequence shown sort of enlarged for these two most common strains of human papillomavirus in which it was possible to design crispr rnas that would uniquely detect one or the other of these viral sequences and the idea here was to implement a protocol where we could take uh human samples these are human anal swabs quick treatment with protein ace k to release the the um in this case double stranded dna this is a dna virus and then a quick isothermal amplification step to amplify relevant sequences followed by detection using the crispr cast 12a protein and again very similar to what i showed you for cast 13 using 404 uh labeled single stranded dnas in this case as the actual detection reporter and so we collaborated with joel palefsky at ucsf who runs a clinic in africa he had collected many thousands of human anal swabs and had access to these in his laboratory he very nicely had already done a lot of work detecting these viral strains using the polymerase chain reaction and so we simply took this crispr cast assay and asked whether we could distinguish between these viral strains and compared the results to those derived from pcr and we were very excited to see that this system worked extremely well not only to detect the virus but also to distinguish between these viral strains so this was really the start of what janice chen ended up deciding to to focus on for for her career at least so far which is to develop this technology as a robust diagnostic for viruses and other kinds of nucleic acid sequences where you would like to be able to distinguish between uh two different types of sequences or more than two um and so in my own lab at berkeley i i was delighted to you know both alexandra eastaletsky with cass 13 and janice chen with cast 12 had done great thesis work and and you know finished their their phds and and had left the laboratory and janice chen founded a company called mammoth biosciences to continue developing the diagnostic work with cast 12. and i had largely you know pivoted my academic lab to other other projects but in the early part of this year when it was clear we were facing a real public health emergency with this stars cov2 pandemic we asked ourselves you know what can we do as a scientist with our expertise to address this emergency and certainly one of the things that we wanted to focus on was testing because it was quite clear and still is that that's a that's an urgent need and um and so we've over the last um you know number of months been working on developing a better diagnostic test using crispr cast proteins and thinking about all of the parameters that are shown here that are very hard to optimize all at once but certainly some of which are uniquely accessible using crispr technology so ideally you would love a diagnostic test to be sensitive to be accurate to be fast and to be cheap and then of course in the current environment thinking about supply chain issues has been uh sort of a repeated challenge as well and so we've been working on this and in the context of a five lab academic consortium that was put together that is links together labs at uc berkeley and at the gladstone and ucsf and so this has been a great opportunity for team science we sort of call it the lab without walls we had initially close to 50 people that were participating in this program really just everybody who had an interest in doing this kind of dropped their original projects and and jumped on board and we set this up as a effectively as running like a little bio tech company within a an academic setting where we had subgroups that were focused on different aspects of the diagnostic to pull it all together as quickly as possible and i want to just show you uh very briefly where that team effort has gotten us and where where we're headed with this and so first of all just a quick update on mammoth biosciences and and um and um i'm a co-founder of this so i want you to know that but i i've been really excited about the work that janice chen has been doing and another former graduate student of mine lucas harrington who together were the primary founders of this company and have really been the leaders on the scientific side in terms of developing crispr-based diagnostics and right now they have this is a slide i i was kindly given by janice chen this shows some of the data from their own research at the company and the comparison here on the right hand side with a number of different commercially available or commercially developed tests for the sars kovi ii virus and you can see that the mammoth kit is looking pretty good in terms of sensitivity and we're actually excited to be able to work with mammoth to provide a beta test center for this test which is going to be available i think in about a month as a laboratory test that can be run on robots why are we able to do that well here's where i owe a huge debt of gratitude to ralph green and colleagues at uc davis because early in this emergency back in the early part of this year at uc berkeley we were able to put up a quick uh you know pop up a a a clinical testing laboratory with a clia license through our university health services and with medical supervision by dr green and so this was very important for allowing us to get testing going in in the berkeley community both on our campus and in our with our community health care partners and more recently we've actually been able to get our own independent clia certification from the state of california and we've recruited a medical director dr petrus john kopolis who's taken over for dr greene and now manages our medical testing that we're doing at the at the lab because of the access now to this medical laboratory and facility we're able to run fda tests we can report a test results back to to individuals and to physicians and we're excited to test the the crispr diagnostic in this context so that we can do side-by-side comparisons with a robust pcr assay to see just how good these crispr diagnostics really are and of course then work to to improve them as needed in our in our uh in our work with the the academic consortium we've actually focused on the cast 13 proteins so mammoth biosciences is working on cast 12 which is the the enzyme that detects dna uh so with a with an rna virus like sars cov2 we thought well we'd actually like to see if we can get this direct detection of rna using cast 13 working for this system and um we we reasoned that advantages of this would be that it's fast it could be quantitative because it doesn't require a separate amplification step in principle and the the target sequence or converting it into dna before detection and it's simple and so we put together this academic consortium five laboratories our our group plus the groups of dan fletcher who's a in bioengineering patrick shu who's bioengineering and molecular and cell biology melanie ott who runs a lab at the gladstone institutes and dave savage in in molecular and cell biology so this has been a really fun uh group of diverse experts who kind of jumped on board and put together this team very quickly this idea of a lab without walls and right now we're working on developing what we call a point of care test that would be useful on site at berkeley and at the gladstone and if it works out there of course we'd love to see it useful at other institutions as well and this is all being done with a non-profit focus the idea is simply to make these tests and make them available to those that would need them at cost and to provide what i consider to be an alternative for surveillance testing for testing asymptomatic individuals to catch those that might be inadvertently infected and spreading virus without recognizing that they're that they're that they are infected so i'm going to describe three different uh formats of this test that we're that we're using right now and we're pretty excited because these have all come together in parallel and are being worked on by different subsets of this large team and the first format is is in a way the simplest and the idea here is direct detection with cast 13 and the strategy is to take cast 13 proteins that are programmed with different crispr rnas designed to recognize a number of different sequences within the sars cov2 genome and then to combine those into a sort of a one pot assay where activation of these enzymes leads to reporter cleavage and release of a signal that can then be detected in a sort of a desktop very simple type of detection device and this is where the fletcher lab comes in because they've been working for a long time on how to do this kind of detection and they have a cell phone based mechanism of doing so that we're implementing right now and for these assays and i just wanted to show you a little bit of these data so this is work done by paranas bozoni who's a postdoc with melaniatt and they just posted a paper on a med archive if you're interested in the details here but bottom line is that we found quite early on that combining multiple guide rnas was an effective way to increase both the speed and the sensitivity of this type of detection and you can see an example of that here where including just one or another crispr rna number two or number four gives a certain rate of fluorescence detection above background but by combining the two in these tests we could get a much faster uh release of the signal and then by comparing doing these lod types of experiments and looking at um numbers of copies that could be detected per microliter in these tests that were typically somewhere between a hundred and a thousand copies per microliter and so this is somewhere between a hundred and a thousand uh full less sensitive than what uh the best pcr tests are typically detecting so uh potentially appropriate for detection of uh of highly infectious individuals and in fact melania has been using samples from the biohub at ucsf to show that you can definitely use the system to detect the virus in samples from highly infectious individuals but not a test that would be appropriate for detecting very low levels of virus so the the and fletcher team is working right now to implement this in a point of care device and uh uh meanwhile we've been also investigating two other formats for crispr tests and the second one is shown here and this has been primarily work led by patrick shu and the group working with him and the idea here is to implement a a quick strategy for amplifying the target sequence and then turning it back into rna using an isothermal strategy called lamp and briefly the way this works is that one takes a sample in this case we're using saliva and it goes through a quick uh lysis procedure this is typically just heat and and protease and then um subjects the sample to this lamp procedure in which the rna target can be quickly converted into dna and amplified and importantly the conversion includes t7 promoters that provide for the ability to convert this back into rna afterwards using t7 rna polymerase now one of the challenges with lamp and you can see it sort of in this plot shown up here is that it's prone to um to signal that comes up independent of the template and um it's fairly well separated in this example but in some cases the signal can come up quite quickly even in the absence of a template and so for um you know for uh commercial tests that are using lamp this is one of the challenges that those tests have is that they can lead to false positives and so this is where the cast 13 detection enzyme comes in and provides a really nice advantage because by turning this amplified dna back into rna with t7 transcription and then using the cas13 enzyme for specificity and using it to detect only those rnas that actually have actually contained the targeted viral sequence it's been possible to improve quite dramatically on the specificity of lamp with the addition of cast 13 and so this is what patrick and his group have been doing and the limit of detection they're getting is a little bit better in terms of sensitivity compared to straight up direct detection with cast 13 and so this is a strategy that again we're working on currently to implement in the in the point of care device developed by the fletcher lab and then the third format is um is one that i think you know really i want to give a huge shout out to various members of my own laboratory who were thinking hard about the fundamental biology of these cast 13 systems and they realized that there might be a way to amplify the activity and the signal of cast 13 by taking advantage of a natural mechanism in bacteria that does that and i'll show you this very briefly so this is a cartoon that illustrates what i showed you before for cast 13 so we have transcripts that are being targeted by the crispr rna and cast 13 and that turns on this trans cutting activity and you can start cleaving fluorophore labeled single stranded rnas and that releases a fluorescent signal but it turns out that one can amplify this when in a in a fashion that we call the nuclease chain reaction by hooking it up to another enzyme called csm6 that is a ribonuclease naturally found in some of these cast 13 containing systems in bacteria and briefly the way this works is that in bacteria there is enzymatic production of these very short oligonucleotides that look kind of like this that will bind to an activation site within the csm6 ribonuclease and turn it on and so that then allows the csm6 ribonuclease to also cut rna just as cast 13 is doing so and and so the idea here and this was really something developed by tina liu in my lab and shruti jackenwall was to add what they call a secondary activator in our diagnostic reaction that's designed to be cleaved by cast 13 when it gets turned on and that cleavage event produces the activator for csm6 and then once this enzyme gets turned on it starts cutting rna this enzyme is cutting rna and this enzyme of course can cut more of this and so then you end up making more of the secondary activator that turns on more of these and you can see how it the whole thing kind of snowballs and so this so far has looked really quite promising in terms of giving us a very fast detection and a relatively sensitive limit of detection i'll show you just a couple of data points here so this is using the cast 13 and csm6 for direct the detection of sars kobe 2 rna and in this experiment um if you you know uh basically you don't have to read every line here but i just want to point out that um that we're seeing that we can detect uh down to about 20 atomoler uh in in terms of copies of rna above a background that we get in these reactions and this uh scales very nicely so it's sort of a you know you can see the kinetics of the activation corresponds to the level of initial target molecule that is present in these in these reactions importantly this also highlights a challenge that we have you can see here very obviously which is that there's significant background in these reactions so you know these enzymes are kind of on a hair trigger and so if they you know if they do get going then you know very quickly they do start to cleave the reporter and you can see release of the signal so this is something that we continue to work on is how to limit this background but even in the format that it's in right now we're pretty excited that we're going to be able to detect fairly low levels of rna quite quickly and you might say well maybe it's not that quick if you look at the time scale you know is it taking an hour to get to that answer and that's where the um that's where the the dan fletcher device comes in so we found that by running our samples um with with that device and if you look over here on the on the right hand side i've sort of expanded this part of the graph what you can see is that the device is so sensitive at detecting the kinetics of fluorescence release that we can very quickly start to distinguish between a background signal and what we get for this 20 atomolar sample so we're continuing to work on this and and improve it but we're we're really encouraged that this chemistry seems to be working out reasonably well very very simple to use it's very inexpensive these proteins are trivial to make and you can make them in very high quantities and we're working currently with a company waynamics that focuses on point-of-care testing to develop appropriate microfluidic chips to run these different chemistries and then use the dan fletcher device for detection so the pieces are coming together we hope and our goal certainly is by we hope by the end of this year or by very early in 2021 that we will have some of these prototype devices operational and running under a an irb type mechanism so that we can do studies on site at berkeley and at gladstone with uh actual donated samples we're using both saliva as well as nasal swabs for the inputs here and we'll see how the data compared to the pcr assay that we run in the clinical laboratory and that's it so i really want to just give a huge shout out to an incredible team of people um you can see this picture is taken uh quite recently this was actually taken on on october 7th which was a an exciting day for our lab and um the first time that many of us had been actually together in quite a while together and we're trying to be appropriately masked and distant etc but um i just want to really thank gavin knott brittany thornton dylan dylan thomas jenny connor abby and enrique all of these folks just rolled up their sleeves and jumped in and started working either in the clinical laboratory or to develop the crispr diagnostic even though i don't think in any case was was that their original project they just recognized what needed to be done they just started doing it which i'm very grateful for trudy and tina and noam are three very talented postdocs who have been developing this nucleus chain reaction under the direction of dave savage and and others at berkeley and working with folks in our lab i really want to acknowledge my entire lab and just a wonderful group of people to work with and we're excited to i normally have about 10 to 12 undergraduates doing research in my lab at any one time sadly we're not able to do that right now with the density restrictions at berkeley but we do hope to be able to welcome our undergrads back to the lab in the coming months and then jill banfield at berkeley is an incredible scientist very wonderful colleague to work with on all aspects of fundamental crispr biology as well as a shout out to emmanuel with whom we started the crispr cas9 work now uh many years ago and then i'll just also point out uh thanks to our our funding sources as well as to ad gene that continues to distribute reagents for crispr at cost to non-profits around the world and they've played a huge huge role i would say in really rolling out this technology for everybody's access and i'll stop there and and thank you and happy to take any questions thank you so much dr doudna for that inspiring presentation i'd like now to introduce dr angel hacksu the uc davis school of madison associate dean for translational research and director of the lung biology center and the ralph green professor of pathology and laboratory medicine who will moderate our discussion and answer session thank you from uh dean rasheer it's been um a great uh pleasure uh joining you vice chancellor muhammad dr green and our distinguished guest dr duna it's been a a terrific presentation and i have been reading the the questions while you were talking so i would be able to um combine them and instead of reading them up one by one i think uh it is dr green's turn to ask to start with the questions so um thank you angela um i'm absolutely thrilled and delighted to be here today uh jennifer i mean this has just been fantastic uh and a little did i know back in march of this year when i began my collaboration with you and your group uh amazing group of of of scientists that we would have the opportunity to host you here today in what has been an absolutely spectacular hour thank you so much we'll begin our question and answer session with uh questions from each of my colleagues and some questions that have come in from the audience as well so our first question is from vice chancellor mohamtra uh prasant why don't you kick off yeah thanks ralph um very exciting talk jennifer so let me start with a very general question given the public's general perception about genetic engineering do you think that medical or agricultural applications of genetic engineering will be more readily accepted hmm uh good question present i i have to say strangely enough and i don't know if this would surprise you or not but i actually think medical uses will be more easily accepted than agricultural and and i it's been a very interesting thing i you know i didn't i didn't know very much about you know these these sort of ethical discussions before diving into this over the last few years but it's clear that there's kind of an emotional i think reaction to um you know thinking about food being manipulated in ways that people maybe don't understand or don't trust or feel are being done for you know commercial reasons that uh that i think has really been challenging for the whole um you know sort of agricultural applications of genome editing and that includes for crispr that uh in in an interesting way are viewed differently when turned to biomedical uses where i think people feel in many cases feel desperate for you know treatments for their kids especially feel maybe at least in some cases more trusting of the process of testing and you know review and and safety uh considerations for clinical uses so that's how i kind of have seen things evolving it's it's very very interesting i that surprised me honestly yeah thank you thank you dr dudna the next question is from dean brashear you've been riley recognized as a pioneer in scientific discovery and as a role model for women and aspiring young people to achieve great success what advice would you give young women entering the stem fields um well my my i guess my advice is is summarized as go for it uh you know i grew up in a in a small town in hawaii and nobody in my family was a scientist and so you know they were they were teachers um lawyers and preachers um so i was lucky that i had you know my parents were certainly supportive of my interests but i was very lucky that i had terrific high school teachers in chemistry and biology in my local public high school hilo high school and then later in college and in graduate school i had you know along the way people who were really supportive and encouraging at moments when i needed it and you know by the way for all the students listening um things haven't always been smooth sailing you know as they never are right you know there's lots of experiments that don't work out or you know all kinds of things that that don't go as you you hoped or predicted and that's when you really need good mentors so i that's my other piece of advice is to really look for uh supportive mentors and they can be anybody men women you know it doesn't matter right it's really just people who are going to see your passion and support you at times when you need that thank you very much so um there were um i know that you gave a wonderful uh presentation about the diagnostic capabilities of the crisper cast technology but there were a lot of questions um in regards to whether there would be um applicabilities for treatment especially for uh treatment and infectious diseases like cov19 for example and would you care to to comment on this just to speculate right well these are great questions because a priori you might think well crispr is an immune system after all so why can't we use it that way right now in humans and um and i think that that um um in principle one could i think the challenge comes back to something i mentioned in the talk which is delivery so you know being able to introduce the the crispr molecules into cells where it's needed um is you know it's a challenge for therapies including for acute therapies like treating a virus and so in the case of coronavirus how you would be able to get these molecules into you know lung epithelia for example is you know it's a non-trivial problem so i personally feel that at least for for this pandemic our best our best bet with crispr is probably using it for detection and um but who knows you know going forward i think you know certainly as as delivery strategies continue to advance and develop it may become possible in the future to have uh you know some mechanism of effective and efficient delivery to cells that would make it a useful therapy i just i think it's unlikely to happen for this pandemic so another question um angelo uh if i may i just want to put another question to you jennifer and that is you've shown us very nicely that the technique techniques of crispr and and all of its uh related methods that you've developed to detect covet 19 viral rna has enabled the development of very rapid specific detection methods of the virus in perhaps less than an hour as a person who's uh involved in diagnostic medicine i ask you does if this if this is applicable to any pathogen will this approach potentially revolutionize diagnostic methods in general i think it i think it could i mean i i you know i would never want to um i i wouldn't want to use hyperbole here but uh but i think i think it could because what's interesting about it is that it takes advantage of what crispr naturally is good at doing which is detection that's the way it functions actually you know in bacteria after all and furthermore it's it's also a system that is naturally multiplexed meaning that it naturally has multiple guides that are you know searching the cell for for different sequences at any one time and so using it that way in a diagnostic setting of course also provides for various advantages and you know including sensitivity and precision as well as detecting potentially multiple different viruses in a in a single sample so i'm personally very excited about this i think it ha it does have a lot of potential um and um and and i think we'll we'll know over the coming months how well it performs in in the real world and that's that's really where the rubber hits the road i think it's one thing to have it working well in the laboratory which many groups have now shown uh can be done and so that's great but now we really need to see how does it perform in uh actual clinical settings where we have patient samples you know better than anyone you know how how variable those can be and um and just some of the challenges with with that so um i'm i'm excited about it but i'm i'm cautious thank you uh back to the the treatment possibilities there were questions regarding sickle cell anemia which is monogenic disease and people were interested in knowing whether in polygenic diseases there might be a possibility and somewhat related to this are the the observed side effects of um of utilization of this technique on off-site um um irrelevant uh gene targeting uh would you care to to comment about this and and what are the um the the prospects of of developing um this technology um for for treatment and what are the um the the best uh target diseases that maybe we can hope for uh being affected right well these are yeah these are really important questions and so the first one first point about multi-genic disease i think they're um it'll clearly be harder to to do to uh you know to implement crispr but but i think i think it's coming i really do i think you know we continue to see advances in the efficiency of the technology um and um already certainly in laboratory settings one can do multiplexed uh genome editing with with relatively high efficiency so i think that's very interesting we're just completing a study right now with the lab of alex marston at ucsf where we've been doing um what i would call a kind of you know single step car t generation where you can knock out a couple of genes and then introduce a gene sequence somewhere else in the genome in one shot with appropriately designed crispr reagents so that's you know very interesting for future use for cell therapies um and so that's uh you know the kind of thing that i think will be my you know more widely implemented going forward as as it becomes possible to um you know again to deliver these molecules efficiently into different cell types and then that of course brings along with it the question about off target and that's your second question and and i think there uh the field you know over the last eight years has you know really dove into this question in some depth and and certainly we know that if uh you know if guide rnas are designed that are have close matches to uh off target positions in the genome that certainly you know one can observe uh what i would call off target editing but on the flip side if uh if guide rnas are designed very carefully and and to avoid those kinds of close matches at undesired positions and if the genome editing molecules are used in limiting amounts then actually off targets are can be quite rare so i don't personally see it as a as a current bottleneck in the field but i just think it's something very important to pay attention to especially for clinical use do you have just very quickly any comments on the uh sorry uh uh dr kent lloyd this has been asking if you saw the news about the ugly lab um results at columbia university today and if you could comment um briefly on this yeah i right i mean i yes i did i did take a look at that work um and you know my view of it is look i mean i think i think consistent with what we've seen from other laboratories uh you can use crispr in ways where you can observe these kinds of undesired um outcomes of editing and and certainly in embryos you know this is a this is a setting where i think there's still a lot to be understood just about fundamentals of dna repair quite frankly from what i've seen um so you know does this does this make me worried does this make me question the whole technology no it just i think it underscores what we're already aware of which is that you know using it in embryos is uh you know is challenging for sure and is going to require a lot more a lot more research to really understand how to use it safely if that were to be done in the future uh jennifer a question i was going to ask you that you've in essence partially answered already which is you know the crispr cass mechanism is is quite incredible really in terms of its precision and and my question was going to be is this just the tip of an iceberg are there other gene editing mechanisms present in prokaryotic cells that we can use you you've indicated you know the cast 12a and cast 13 it's all part of a sort of general toolbox do you see this thing developing in time into an industry almost of of various uh gene editing mechanisms that we can use from bacteria oh absolutely and i think i think we're already seeing that i mean there you know there are certainly are there's a whole sort of panel of companies that have been set up to mine bacterial genomes for these sorts of things these sorts of systems and enzymes and then of course many many academic labs that are doing that as well and and and not only mining what's found in nature but continuing to engineer things based on what nature has done to you know make those proteins do things they maybe don't naturally do so yeah i i agree with you 100 i think i think we're going to see i sort of imagine this yeah this whole kind of you know uh um you know virtual toolbox that's going to be available and um and in the future when some kind of genetic manipulation is desired one will be able to go to that virtual tool box and you know pull off the shelf the appropriate tool to do the job great thank you angela i'm going to hand back over to you because i think we're reaching the end we could carry on forever here right yes but but we have two more minutes so um if possible um renee solis is asking um how is it known how the crisper dna gets into the bacterial genome um genomic crispr array so quickly before the fast lysis occurs yeah that that that is a that is a really important fundamental question so um a lot is known about the crispr integrase itself and that kind of that whole reaction mechanism but what still not really known very very well is is is how it works in the context of the life cycle of the virus right because as this questioner is pointing out you know viral infection especially in bacteria very fast right especially for a lytic virus you know maybe 20 minutes right it's quick so would there really be time to integrate the sequence make the rna make the surveillance complex and then go probably not so i think the best data that i've seen in the field right now for this is that um you know in bacteria a lot of these these bacteriophage are maybe not a lot but you know there's a significant percentage of those phage that are defective phage and so what's thought to happen is that in a you know bacterial population some lucky cells get received these defective phage and so they are able to acquire functional spacer sequences in the crispr array and then provide protection to those cells and so in a bacterial population you know if a lot of the cells are killed off but you have some survivors and they pass on their immunity then that's fine so that's that's what we think happens in a natural setting with crispr thank you so much i'm going to turn it back to to dr mahapachana for closing remarks thank you so much uh jennifer for being with us today and sharing your valuable time as well as expertise and once again congratulations for all your remarkable achievements and contributions to the human health and the society in general i would also like to take this opportunity to thank dean brazier dr green dr hakshu and to all our audience who are joining us today enjoy the rest of the day [Music] thanks

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Length: 73min 4sec (4384 seconds)

Published: Thu Nov 05 2020