Nobel Lecture: Jennifer Doudna, Nobel Prize in Chemistry 2020

jennifer downer was born in 1964 in washington d.c in 1989 she received her phd from harvard medical school in boston she currently serves as a professor at university of california berkeley in the u.s jennifer doudna i now welcome you on to the stage we are very much looking forward hearing your lecture i'd like to begin by thanking the royal swedish academy of sciences the nobel prize committee in chemistry my family including my spouse jamie kate our son andy my sisters ellen and sarah doudna my friends colleagues and of course my former and current lab members about whose research i will be speaking today it's a wonderful honor to have this opportunity to share with you the science that we've done over the last few years and to discuss the extraordinary opportunities and exciting advances that are happening right now with crispr cas9 as a genome editing technology i thought i would begin by telling you about the origin of the ideas around crispr and this began at least two decades ago with research in microbiology laboratories showing that bacteria might have an adaptive immune system a way to provide protection against viral infection that would allow cells to acquire it in real time and then protect the cells from those viruses using this recording system in the genome and this system came to be called crispr and as i'm showing here this is a mechanism by which bacteria and our kale cells can adapt to viruses by integrating small pieces of viral dna into the organism genome at a site called crispr and then using that information to make an rna copy of the sequence that can then provide the information required for detection and cleavage of viral dna or sometimes viral rna using crispr associated or cast proteins and this slide shows the way this system is thought to act in bacteria and represents experiments that were done in the field early on both using bioinformatics and molecular genetics to understand the function of this pathway this video shows the way that we imagine crispr operates in nature where bacteria that might be growing in a biofilm are being infected by viruses and when the virus injects its dna into the cell the cell can acquire a small piece of that foreign dna into the crispr locus a place in the genome that consists of repeated sequences that flank these integrated sequences from viruses that are called spacers and the cell then makes a copy of that integrated sequence in the form of rna which is then processed into individual units that include a sequence derived from a virus those rnas combine with a second type of rna called tracer and a protein called cas9 to form a surveillance complex an rna guided protein that can search the cell looking for sequences that match the sequence of the guide rna and when a match occurs the crispr casts protein cas9 is able to cut double-stranded dna triggering destruction of the dna in a bacterial cell and this pathway has been operating for a long time over evolutionary time evolving in bacteria and has over time been diversified into many different forms of crispr systems that can operate effectively in these organisms but today i'm actually going to talk about in particular about one type of crispr caste system that uses the protein cas9 as i showed in that example this slide shows the three steps in crispr acquired immunity that include adaptation expression and interference and in the research that i did in my laboratory with a number of my former students beginning with blake wiedenheft and then rachel horowitz and folks that came after them we began investigating in particular this third step in the pathway called interference the stage of the crispr pathway that involves an rna guided detection of viral dna back in 2011 we had the good fortune to begin a collaboration with emmanuel sharpentier and her student christophe chielinski and this launched a wonderful opportunity to answer what was at the time a very interesting and intriguing question in the crispr field namely what is the function of the protein called crispr cas9 and we were fascinated by this question because this particular protein had been implicated in protection of cells and in particular a type of bacteria that emanuel's group was studying called streptococcus pyogenes an organism that infects human beings and this particular organism has a crispr cas9 protein encoded in its crispr system that was implicated in protection of cells from viral infection but the question was how and so in our collaboration we investigated this and addressed the question by doing biochemical experiments that involved working with purified crispr cas9 protein and the rna that guides it to target dna sequences in cells and what we found and this is research done by martin yinnick a former postdoc in my laboratory and chris tylenski working in emmanuel's laboratory is that crispr cas9 in nature is a dual rna guided protein as i showed in that introductory video this is a protein that uses a crispr rna molecule to direct it to a sequence of dna that matches the crispr rna sequence and it also requires a second rna molecule called tracer that provides the an interaction with the crispr rna required for assembly with cas9 and so together these two rnas guide cas9 to dna sequences where the protein cas9 is able to make a cut in the double helix of dna i think one of the wonderful aspects of crispr and this project with emmanuel's group is that this was the point in our research where what began as a curiosity driven investigation morphed into a project that had much broader implications because once we understood how nature uses a dual rna system to guide cas9 to target dna sequences it was possible to engineer the dual rna guide as a single guide rna shown here that included both the targeting information and the structural requirement for assembly with the cas9 protein in a single rna molecule and once this experiment was done by martin and by kristoff we found that cas9 could be programmed with single guide rnas and directed to cleave double-stranded dna at a desired sequence taking advantage both of the actual targeting information in the crispr rna molecule as well as the requirement for what's called a pam sequence a proto-spacer adjacent motif pam is easier that occurs right next to the target sequence in the dna and those two elements are required for cas9 to recognize and cleave dna but this this uh this requirement is sufficient for cas9 to function as a an rna guided dna cleaver and i wanted to show you a key experiment that martin yanek did to test this idea of a single guided cas9 protein that could cleave different sequences of dna and the experiment was to design guide rnas that would recognize several different sequences in a plasmid dna a circular piece of double-stranded dna that we could purify in the laboratory and what you're seeing on the slide here are marked in red five different sites in this plasmid dna that were chosen as target sequences for single guide rnas that we produced in the laboratory and martin did the experiment of taking those single guide rnas adding them to the purified cas9 protein and then incubating together with the plasmid dna molecule in a laboratory test tube and then to analyze the result of that experiment he separated the cleaved dna products an agarose gel system which is shown here which is simply a way of separating dna of different sizes from from each other and what you can see in each lane of this gel system is that depending on where the guide rna was directed to to interact with the plasmid dna cas9 would generate a cut and then by also cutting the plasmid at a separate place so that two double-stranded breaks were introduced into the plasmid at one time we could release these little fragments of dna that i hope you can see on this gel system that migrate at different positions representing different sizes of these dna fragments and i have to say that on the day that martin yanek did this experiment and got this result we were just incredibly excited it was the pure joy of discovery at recognizing that we not only understood how this bacterial enzyme cas9 functions but we had actually figured out how to engineer it as a simple two-component system for directing dna double-stranded cutting now why was that so exciting well it was of course interesting to know that we could we could harness our knowledge in this way and engineer the protein to to have this desired cleavage capability but in addition it also allowed us to imagine how crispr cas9 could actually be harnessed as a technology for something quite different in eukaryotic cells namely cells like plant animal human cells all of which treat double strand breaks in dna differently than the way they are treated in bacteria and i'll show that on the next couple of slides so in eukaryotic cells when the cell receives or detects a double-stranded break to the genome to the dna in the cell the break is detected and repaired typically before it can cause cell death and the repair pathways involve either a non-homologous end joining event shown on the left side of this slide that can sometimes introduce a small disruption to the dna sequence or there can be an integration of dna that has homology to the sequence of dna flanking the double-stranded break and in that case a new piece of genetic information is incorporated into the genome at the site of the original break this was research that had been done over the previous couple of decades before emmanuel and i did our work with cas9 but we recognized that the activity of cas9 its ability to introduce a double-stranded break to dna at a desired position by directing it with these single guide rnas could allow scientists to introduce double-stranded breaks into a genome that could trigger the kind of repair that i'm showing here and so we imagine the system working very much like in this video where the cas9 protein could be directed to enter the nucleus of a eukaryotic cell and with its guide rna search the genome for a 20 base pair sequence that would match the 20 nucleotides the 20 letters of the guide rna that would provide a match and when that match occurs we now understand that cas9 is able to unwind the dna it allows the cas9 protein to generate a precise double-stranded break in the dna by cleaving each strand of dna and then those broken ends are handed off to repair enzymes in eukaryotic cells that lead to dna repair and in the process of of repairing the dna there is the ability to introduce a change to the genome at a precise place which is really the definition of genome engineering so on the next few slides i want to share a few things that we've learned over the last few years about how castline is able to achieve this kind of of editing in genomes of cells by triggering double-stranded breaks and i'll start with showing a molecular model of the cas9 protein and this is based on a crystallographic structure that was solved originally by several different laboratories our own the lab of martin yannick the lab of osamu nureki and subsequently many others who have contributed to understanding the actual molecular basis for cas9's function and in this slide what you can see is the white protein cas9 holding on to its orange guide rna and a blue double helical dna molecule and i'd like to point out that in this structure we can see the mechanism by which cas9 uses an rna guide to interact with dna at a precise position because you can see the orange and blue helix formed inside the cas9 protein representing the interaction between the guide rna and one strand of the target dna molecule this is the actual way that these proteins are able to find and hold on to dna prior to dna cleavage the next thing that we learned in the lab in studying how this cleavage event actually works is that a series of experiments showed that cas9 is a highly dynamic protein it has to be to be able to handle dna unwind a double helix the way we know it does it has to be able to move and through a series of of chemical experiments that allowed us to detect motions in different parts of the protein we discovered that cas9 has undergoes a large conformational change as it holds on to dna and catalyzes cutting and i'll show that in this video here so this shows the casino protein alone morphing to the structure it forms when it binds to the guide rna which you can see now in orange this is the the structure that is able to search the cell looking for a matching sequence of dna and when that match occurs there's an additional structural change in the protein that accommodates the dna molecule so that you can now see the rna dna helix forming inside of cas9 and then finally this yellow part of cas9 swings into position so that it can cleave the dna strand that is attached to the guide rna and this is a very important aspect of the the chemistry of cas9 catalyzed dna cutting because it provides a mechanism for sensing the interaction between the guide rna and the dna and ensuring accuracy of castline's cutting mechanism in work that was done just over the last few years fugo jong a former postdoc in my laboratory and and very sadly now deceased did a series of very exciting structural experiments to reveal the shape of the cas9 protein when it's engaged on a full-length dna molecule this shows again the guide rna in orange the dna strands are in blue and magenta and you can see how the dna is held open by the cas9 protein and allowed to interact with the cleavage sites in the enzyme for precise double-stranded cutting of both strands of the dna and then in this image you can see in green the one of the cutting parts of the enzyme swinging into position so they can actually catalyze the chemistry required for dna strand break and then i wanted to also point out that in addition to these conformational changes that happen in the cas9 protein itself we also now understand that this protein is quite dynamic in the way that it interacts with long pieces of dna for example chromosomes in cells it has to be able to move very quickly along the length of dna searching for a sequence that will bear a complementary match to the guide rna how does that work it seemed like an extraordinary capability to us initially and so in experiments that we did in the laboratory over several years with a number of former lab members and collaborators we came up with the model shown here that i think is consistent with current data suggesting that the cas9 protein has the ability to bind and release dna very quickly and that allows it to search through very large very really vast stretches of dna quite fast to allow interrogation of the the sequence that's being searched for a match with the guide rna and so in this example the guide rna is in red and you can see that our data suggests that when this cast 9 protein with its guide interacts with dna it begins to pry apart the two strands of the double helix to allow the protein to ask is there complementarity for binding to the crispr rna and if there is then our data suggests that the strands of the dna continue to melt apart that allows the rna dna helix to form inside the cas9 protein if that helix is perfect or close to perfect then the enzyme is triggered to cleave dna and this is quite an amazing mechanism and clearly allows bacteria to search the cell very quickly looking for viral dnas to destroy but in eukaryotic cells this mechanism is equally effective at triggering double-stranded breaks that can be repaired and trigger changes in the format of genome editing that we now understand can be effectively catalyzed by cas9 so in the next part of the talk i really want to turn my attention to where this technology is going and there's a lot that one could say here so i'm really going to hit on a few of the highlights and first of all is the fact that genome editing extends across all of biology it can be used for fundamental research but also for exciting applications in public health in agriculture and in biomedicine and it's very important also i think to point out that genome editing can be conducted in many different kinds of cells and fundamentally in the two kinds of cells that i'm pointing out here one category of cell is a somatic cell that's a cell that is fully differentiated it does not have the ability to create a new organism versus a germ cell which is a cell such as a sperm or an egg cell or cells in an early embryo that have pluripotency and they're able to differentiate into many different cell types as an organism is forming if if genome edits are create are introduced in a somatic cell those changes to dna are not heritable so they affect only one cell or one tissue type or one one individual organism but if genome edits are introduced into a germ cell they have the potential to be heritable and to introduce changes that become part of not only an individual but all of that individual's progeny and this is of course very powerful when we think about using it in plants or using it to create better animal models of human disease for example as has been done using crispr cas9 in mice and rats it's very different when we think about how it could be impactful in in human biology and the of course enormous ethical and societal issues that are raised by the possibility of using germline editing in humans and i won't say too much more about that but there's a it's been become a very active area of my own work over the last few years is to think about responsible use of crispr cas9 and in particular its use in humans and ensuring that there's transparency and careful thought that goes into work particularly when applied in the human germline but in somatic cell editing i think there are extraordinary and exciting opportunities that we will will be developed in the near future and one of them is in the area of correction to disease-causing mutations in humans and this is just one of many examples the opportunity to correct a mutation well-defined that causes sickle cell anemia and can now be corrected using crispr cas9 technology and this is no longer a potential opportunity but it's actually been realized by using crispr cas9 to correct the the disease-causing mutation in a patient and to show that this technology is safe and effective for this kind of treatment of genetic disease i think this is a very exciting way to imagine how this technology will impact human health in the future and of course with germline editing one has to think about how this technology will advance in the future and i i certainly imagine that we will see increasing applications in germ cells including in the human germline and of course that has to be managed very carefully and i'm pleased that there's been an active international effort to control the use of crispr cas9 and certainly to encourage transparency especially with the recent release of a report that discusses the science and the technology around human germline editing and very importantly establishes criteria for using crispr cas9 in the human germline in the future in the last few minutes i'd like to turn to where crispr technology is headed in the future and one of the aspects that of this work that's fascinating is the incredible diversity of crispr systems in nature this continues to drive the field in terms of fundamental biology understanding what these systems do in their natural settings and microbes and of course how they may be harnessed as technologies and other organisms as was the case for crispr cast 9. i wanted to mention briefly the effort to investigate new crispr caste systems and one of the recent findings that we've had with our collaborator jillian banfield at berkeley is the discovery that phage bacteriophage the viruses that bacteria use crispr to to protect against in fact can also carry around their own crispr cast systems and this is one example shown here a protein that we we named crispr caspi that is entirely phage encoded it's a tiny protein but it nonetheless has the rna guided dna cutting capabilities that we discovered originally in crispr cas9 and so this is a i think a fascinating example of nature's diversity as well as the opportunities that this protein might provide for future applications including in cases where one could benefit from having a very tiny protein a small gene that encodes that protein that could be potentially more easily delivered into eukaryotic cells we've also been very interested in other biochemical activities of crispr cast proteins and i'll mention here very briefly that in research done originally by alexandra east soletsky a former graduate student in my laboratory and mitch o'connell working in partnership with her they discovered that a class of crispr proteins called cast 13 which are naturally rna targeting enzymes have a biochemical activity that could be harnessed as a detection mechanism and this was an experiment done originally by alexandra e salewski and actually suggested by jamie kate the idea of putting fluorophores onto small pieces of rna that were cleaved upon cast 13's detection of an rna sequence using its rna guide and this system turned out to be highly effective at detection of rna molecules all the way down to about picomolar levels this was in just very early experiments with an off-the-shelf kit called rnase alert in the laboratory but it also triggered our interest in the biochemical activities of other families of crispr cast proteins and in research done a couple of years ago janice chen a student in my lab at the time showed that another family of enzymes called cast 12 also have the ability to cleave single-stranded molecules in this case single-stranded dna upon recognition of a double-stranded dna target and in this experiment what janice chen showed is that the cas-12 protein upon recognition of a double-stranded dna is able to cleave single-stranded molecules of dna shown in this experiment in the panel in the center of the slide showing very rapid degradation of a circular single stranded dna molecule in this experiment which is an activity of biochemical activity that we do not detect for cast nine and working with a colleague joel palefsky at university of california san francisco we were able to use this cast 12 activity for detection of the human papillomavirus in human patient samples and even to distinguish between two different strains of the human papilloma virus which is what's shown in the panel in the center of the slide and so this told us that not only could crispr cast enzymes be useful for detection but they could also be useful for specificity in figuring out what type of viral signal might be present in a patient's sample and of course in the current sars cov2 pandemic that has become a worldwide human health issue we and others have been using this type of crispr cast detection to identify samples in in patient saliva or impatient nasal swabs and to do so very quickly ultimately we hope with a readout that will involve a simple mechanism with a cell phone that can record the results of these diagnostic tests and help people everywhere to screen themselves against this virus and importantly provide a mechanism for future pandemic preparedness because of the programmability of the crispr cast enzymes which is a fundamental property of their biology and their ability to be harnessed as technology so i'd like to to close by just pointing out that the rna guided gene regulation that we observe in crispr caste systems is fundamental to the way they work in bacteria but also to the way they operate as technologies for genome editing and beyond delivery and control are going to be key in the future so we need to have better ways to deliver the crispr cas9 and related proteins into cell types of interest for genome editing including into human patients and fundamental research will continue to drive the field forward as it has in the past and has it will in the future the possibilities are endless and i'm excited about what will happen in the future both with fundamental research and with the applications that will solve real-world problems in human health and the environment i'd like to thank my past and current lab members with whom i've had the joy of doing science together over many years i want to thank the many colleagues who have been involved in the fields of genome engineering in dna repair and in the applications of genome editing that have made the field so exciting over the last few years and finally i want to thank my colleagues at the university of california berkeley who i've had the joy of working with and where we share together the dedication to public education and research that makes our work so rewarding thank you very much

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Length: 31min 57sec (1917 seconds)

Published: Tue Dec 08 2020