Scientist Stories: Jennifer Doudna, The Code Breaker

Welcome to Director's Dialogues, our virtual conversation with visionary leaders of science, healthcare, and biotechnology. These dialogues offer an inside perspective on the emerging opportunities, challenges, and trends in biomedicine, education, public health, and related fields. Our guests share their most meaningful experiences and observations, tell us about the opportunities that most excite them, the challenges that most concern them, and the innovations that could dramatically enhance our collective ability to address those challenges. You can participate in the discussion as well by submitting questions through the question and answer box at the bottom of the screen. Send your questions at any time during the discussion, and we'll answer as many of them as possible. Tonight, we're discussing one of the most exciting and promising areas of biomedical research today, the gene editing approach broadly known as CRISPR. And we have the perfect person to guide us in that discussion, Jennifer Doudna, who won the 2020 Nobel Prize in Chemistry for her role in developing the CRISPR-Cas9 genome engineering technology, and her lab is worldwide— labs worldwide have eagerly incorporated this new tool, creating a CRISPR revolution with huge implications across biology, medicine, agricultural genomics. Jennifer is also a leader in public discussion of the ethical implications of genome editing. Jennifer is the Lee Ka-Shing Chancellor's Chair and Professor of Chemistry and of Molecular and Cell Biology at the University of California, Berkeley. She's also an investigator with the Howard Hughes Medical Institute. In 2014, she and now Whitehead Institute member Jonathan Weissman co-founded, and she is now the president of the nonprofit Innovative Genomics Institute, which seeks to drive development of genome engineering as a powerful tool to treat human diseases and end hunger. She is president of the Innovative Genomics Institute and also co-founded several companies created to bring Casper-based technology to the market. Jennifer earned a bachelor's degree in chemistry from Pomona College and a PhD in biological chemistry and molecular pharmacology at Harvard, with Jack Shostak, also a Nobel laureate. After completing a postdoctoral fellowship at the University of Colorado, she joined Yale faculty in 1994, and in 2002, she joined the Berkeley faculty where she has been since. She's a member of both the National Academy of Sciences and the American— the National Academy of Medicine, and has won a slew of prestigious awards beyond the Nobel, and please forgive me, Jennifer, if I don't list them all here now. And finally, Jennifer's life, work, and impact is the subject of Walter Isaacson's recent biography, The Codebreaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race. Jennifer, thank you so much for joining us tonight. So let's start.

Wonderful to be here. Thank you for inviting me, Ruth.

Thank you. Thank you. It's great. Um, well, Let's start by focusing our discussion on, of course, CRISPR, the achievement for which you are most widely known. But before CRISPR, you had a stellar career studying structural biology and RNA. Could you tell us a bit about that work and how you made the transition to your current research, how it got to where you are today?

Well, as you mentioned, I got into science from a chemistry perspective. And when I came to graduate school in Boston, I knew absolutely nothing about biology for the most part. I was fortunate to join the lab of Jack Szostak right across the river from MIT, where I started working on, of all things, the origin of life. And the question we were asking in the lab at the time was the role of RNA molecules in establishing a self-replicating system— in other words, a system whereby simple cells could maintain genetic information and copy it using the very same molecule, namely RNA. So I, you know, that was kind of the beginning of my, my career, and in a way I never left it in the sense that I fell in love with the study of RNA. It was such a fascinating area of biology, and all of the projects we worked on over the years have really focused on the role of RNA in modern biology, including RNA molecules that function like enzymes. We call them ribozymes, and I worked on structures of ribozymes early in my career, to looking at how RNA molecules are involved in host-pathogen interactions, viruses that infect human cells, for example. And that actually led us eventually to CRISPR, which, as we'll get into, is an RNA-programmed system that bacteria use to fight infection.

Yeah, so why don't you give us a quick primer? And, you know, I have a feeling that a lot of people who are listening do know what CRISPR stands for, but let's start at the beginning to include everybody.

Well, it's, you know, it's kind of a catchy acronym. It stands for Clusters of Regularly Interspaced Short Palindromic Repeats. Oh, don't make me say that again. And what it means, what it symbolizes, CRISPR is an immune system in bacteria that uses sequences of DNA that come initially from viruses that are transcribed into molecules of RNA that can use a search and destroy mechanism in cells to find and cut up virus genetic material. And that's fundamentally what CRISPR does. It's an adaptive immune system that relies on RNA to find and ultimately destroy viruses in bacteria. Bacteria. So we started investigating it because I was frankly fascinated by the idea, which was a hypothesis at the time, that bacteria might have an adaptive immune system, something that prior to work on CRISPR was really thought to be confined to eukaryotic cell types. So that fascinated me. And furthermore, the idea that this would be a system driven fundamentally by RNA molecules was I thought, you know, really intriguing from a biology perspective. So we started investigating it from the, you know, just a curiosity-driven motivation, but it became clear pretty quickly that the system was set up in a way that could easily be harnessed as a technology. And that's really what CRISPR has become famous for, is that it's now widely used to make changes, targeted changes, to the DNA of cells in a way that allows scientists to understand the function of genes, but also importantly, to correct disease-causing mutations.

Yeah, so, so you just mentioned it, it came, it started with a very basic observation, a curiosity. So, so, so tell us a bit more what the discovery and development of CRISPR tells us about the relationship with basic and applied science research, because that wasn't your intention initially, I assume.

Right. I started working on CRISPR as a, you know, in our, with our interest in this RNA-driven immune system, and I quickly got connected to Emmanuelle Charpentier, our collaborator on the CRISPR-Cas9 enzyme protein that cuts DNA molecules using an RNA guide. And that research for both of us began as a collaboration to understand the chemical mechanism of RNA-guided DNA cutting, which is fundamentally what the CRISPR-Cas9 protein is capable of doing. And, you know, I think we both came to that project with a fundamental curiosity about the system, but we also were interested in it as a way to understand more broadly how bacteria are able to fight back against viruses. So it was, you know, it was a a great example of fundamental research that ends up going in a new direction. Namely, once we had the mechanism of Cas9 in hand, it was clear that it could be a very useful system for triggering the kinds of genome editing that many people were interested in, but there wasn't a really easily deployed technology for doing at the time.

Yeah, and then once this discovery came out, you know, the CRISPR research and development has moved so quickly over the last decade. And there's so many applications. So I just want to sort of name a few local examples from Whitehead Institute scientists, like your former colleague and now my colleague, Jonathan Weissman, is using CRISPR technology in many creative ways, developing potential therapeutics for viruses, to tracking ways of how cells repair damage or how you can actually follow cancer origins. And then also at Whitehead, Sebastian Laredo has used CRISPR to do a screening to better understand how dangerous pathogens such as Toxoplasma actually function. What are, in your mind, the most interesting ways that CRISPR is being used today?

Well, I love those examples you just gave because I think they illustrate how CRISPR is being used to fundamentally change our understanding of the genome, namely all of the genes encoded by an organism that make it possible to function as it does and to interact in its environment. So that's, that's clearly one important direction that the CRISPR technology is headed. It's worth pointing out that there are a number of clinical trials that are either in progress or have already announced results showing that CRISPR can be used to correct disease-causing mutations or make changes in patient cells that mitigate disease. And here I'm thinking in particular about sickle cell disease and a liver disease that is, you know, fundamentally a protein aggregating— has a protein aggregation phenotype that can be corrected using CRISPR in the liver. And so, you know, these are, I think, really motivating to all of us in the field because we can see the extraordinary potential of this technology to change the lives of people that otherwise are suffering from untreatable, incurable, genetic diseases. And I also want to point out that there's a huge opportunity also in agriculture to make changes in plants and in the microorganisms that support agricultural product development that I think will be transformative in the future.

Yeah, there's a lot of discussion about whether those plants then are genetically modified because they have no foreign genetic information, right? That's a very interesting discussion.

Yes. As you know, different countries draw different conclusions about that too.

Maybe you can actually eat those tomatoes then in Europe. On the other hand, you were mentioning on a somatic level, on a person level, to restore functions of disease genes, but the extraordinary capabilities of CRISPR also bring up some thorny questions about genome editing and how much it should be used, not just to genome editing, which we just discussed, but actually germ cell editing, and which is changing genes actually in egg and sperm, and thereby making the changes permanent and not just limited to the person., but also then to their descendants. Um, how do you believe that scientists and societies should be approaching such questions, and how has that evolved, that discussion, since you initially brought that concern up when many people were not yet thinking about this, although some people were already starting to work on it? Right.

Well, I think this is an interesting example where science we find ourselves as scientists faced with some ethical challenges and debates that we have to, I feel, grapple with openly. I think first and foremost, rather than running away from those issues or ignoring them, I think we have to be facing them head-on. And to me, that involves an open and transparent conversation that includes not only scientists and clinicians, but also other stakeholders, essentially anyone who has an interest in this, I think, should be invited to be part of it. And, you know, it's a big challenge. How do you, how do you do that? How do you get started with something like that? So in our case, we were fortunate that at the Innovative Genomics Institute back in early 2015, we convened a small meeting that brought together scientists from around the, the U.S. at the time who were working on various applications of CRISPR, including in embryos, or at least thinking about doing that, and as well as some of the leaders of the original Asilomar meeting back in the '70s that had grappled with ethical challenges around molecular cloning, which was at the time a new technology that again raised potential challenges and health risks for humans. And since then, since that 2015 meeting, I've been very pleased pleased that many people have taken up this charge. We've got the National Academies now in multiple countries that are involved. We've got the World Health Organization involved. And so there are now multiple groups that are deeply investigating not only the potential of the technology and its opportunities, but also really looking at appropriate ways to regulate it as the opportunities continue to grow because there's no putting the genie back in the bottle, as they say. And I think that, you know, we just have to, you know, try to work together to ensure responsible use of CRISPR as the field moves forward.

Have governments made real decisions, or is it left to the scientists?

Yeah, not really. I mean, I think it's at this point, it's still really in the hands of scientists and scientific societies. To put forward guidelines that are, you know, being distributed right now among communities of researchers and clinicians that are using CRISPR. And, you know, it'll be interesting to see what happens in the future. I think some people that are attending this talk tonight may be aware that already there's been an announcement 3 years ago about— actually, almost 4 years ago now— about CRISPR being used in human embryos to create a pregnancy of twins who were born with CRISPR-edited genes. And, you know, I think that announcement back at the end of 2018 really was kind of a wake-up call that, you know, this is not something that might happen sometime in the distant future, but it's actually a technology that has that capability now. And so we have to be very vigilant about, you know, how it's going to be utilized in the, you know, around the world.

Do you think the scientific community is strong enough to really put forward restrictions?

Yeah, it's a good question. I mean, I think that's, in a way, it's sort of the best we have. Because I think it's—

The most educated, right? Those are the people who know what the potential is.

I think that's right. It's very difficult. I mean, I've talked to a number of our legislators and legislators in other countries, and for for the most part, they're, you know, I think they're really well-meaning and they want to do the right thing. It's just that if they're not trained as scientists, which most of them are not, they feel out of their, you know, out of their depth in terms of really understanding the technology. So I think, you know, having scientists engaged in this and putting out reports as we've been seeing that, you know, continue to evaluate the technology as it advances and it's moving, you know, the field is moving extraordinarily fast. But I think this is, this is, you know, this is the best we can do in a way, that we really just have to keep continually evaluating where the technology is, what are the opportunities, what are the risks, and what are the sort of costs and benefits of using it in different ways.

Yeah, it's sort of interesting in that regard. So it was really played out very well in Walter Isaacson's The Code Breaker book about the discovery and development of CRISPR, was sort of this example about competition and collaboration, which also in this case, you know, the competition obviously of driving people to push the envelope, right? And both of which have a real good role in driving science forwards. How do you think about balancing competition and collaboration? You have had some lessons about this, and so maybe you can talk about that.

Well, I think you make a great point. I think, you know, we need both, and I think this is true in basically all human endeavors, that we have competition and we have collaboration and they both play a role in advancing science. In my experience, I always try to compete with myself and I'm always asking myself, am I doing the best work that I could be doing? Am I contributing in the way that's going to be most impactful given my expertise and my interests and things? I always try to come back to that. Regarding collaboration, I think that Gosh, I just, I love working with smart people. You know, it's one of the joys of our profession. And so it's been a really exciting— this is something I didn't anticipate when I got into science as a student. I didn't realize what, what an exciting, you know, opportunity it is really to work with other people who bring their own perspectives, their own expertise to bear on a problem, and how incredibly satisfying it can be to grapple with something very challenging that you're trying to figure out, and you have, you know, multiple minds working on it together, and then when you come up with a solution or an answer, it's just, you know, that shared joy of discovery is, you know, it's extraordinary. There's nothing like it.

Yeah, yeah, and I see that very much at Whitehead, and actually having Jonathan Weissman join the Whitehead, you know, He clearly collaborated a lot when he was at UCSF in creative ways of using CRISPR, and now here he expanded this in an interesting way. So for example, Jonathan and Jingke Wang are planning to use gene editing to engineer plants to sequester far more carbon than what they can do. So it's very innovative, and two people from very different directions coming together. And so That's, to me, that's actually the kind of collaboration which works very well when very different expertise comes together. So, so what do you think makes collaborations with colleagues most effective? And also, we really want to be a place to foster the spirit of collaboration, so I'd like also in your answer, you really emphasize the collaboration and, you know, the competition is not a fight with somebody else, but it's actually a competition with yourself. I can very much identify with that. So how do we really foster this kind of spirit and provide the environment, which I'm sure you're also grappling with where you work, that encourages collaborations?

Yeah, it's a great question, Ruth. I think, you know, in my experience, collaborations work well when you have good chemistry between the people, that is. You know, you really have to have people that like to work together, that get along well with each other, that, you know, where the work is fun. You know, it has to be fun. I think it has to be enjoyable. And it also has to involve people bringing different kinds of expertise to a problem. In my experience, collaborations have kind of fizzled when everybody's kind of got the same perspective on something, and especially if the problem or project is not is not a top priority for the group, you know, for people, or for at least some people in the group, then it also doesn't work very well. So you have to have a really compelling problem that all the people participating in are excited about, and that they feel they have a unique piece of expertise or perspective to bring to the project. So they're, you know, their, their work is really necessary to the success of the project. And when those things are true, magic happens, I feel. It can really accelerate the kinds of things that one can get done. And so, yeah, you brought up a great point. How do you foster that? How do you create an environment that really encourages this? And as you know, funding agencies have explored different mechanisms of funding where you try to fund only groups that have that sort of composition, et cetera. I think one of the challenges— and I don't know the right answer to it, but we're trying to think about it here at our institution— Institute how to deal with it is, how do we change the reward system in academic science so that we make sure that we value people that are working effectively in collaborations and maybe are the critical player in some cases in collaborations, and yet, for whatever reason, their name may not end up as the first or the last author on a publication? And how do we grapple with that? How do we grapple with more junior scientists collaborating with somebody who's very senior in their field? And again, figuring out how to ensure that everybody gets credit for the work that we're doing. And I don't have any magical answers to this, but I think there are some creative ideas that are being discussed about— and we already see papers published with consortia listed as the author, where you have a detailed description of what each person person in the consortium contributed to the project, things like that, so I think there's some interesting ideas out there that are being explored to foster this collaborative spirit even more than we're already doing.

Certainly COVID did not help to foster the collaborative spirit. No. So what would be your advice for a young faculty member? You just mentioned young and older in terms collaboration, because that's a question I'm often—

Yeah, well, I encourage our junior faculty to— and we actually have a number of project scientists at the Innovative Genomics Institute as well, kind of analogous to Whitehead Fellows, I would say— and I encourage them to really go after problems that they think are impactful, that they're excited about, that that are really well suited to their expertise, but not to be afraid to pull in others who have complementary things to bring to the project, whether it's a new perspective or a particular expertise they bring to it. And, and, and again, you know, I think it really comes down to ensuring in the end that we value collaborative work when those folks are reviewed for promotions and advancement, that we ensure that we as an institution, that we have a culture here that really does value that kind of collaborative work and not simply looking at where someone's name is on a publication.

So do you think funding— NIH is getting that? Yeah, I think in some cases. So in terms of like your contributions?

I think it's still a challenge, isn't it? Because, and I've been on study sections, I'm sure a number of the faculty, if there are faculty listening to this, you've done this where you're asked to evaluate people that are applying for funding and it can be tricky if they are a person who, let's just take bioinformatics as an interesting field where people are often working collaboratively with a number of groups on maybe a variety of projects where their contributions are critical and yet they may not, for various reasons, end up first or last on publications, and so to evaluate their contributions, you really in a way have to read the papers and you have to use other metrics to understand what their contributions are, so I think we're still grappling with that, but I think the NIH is Increasingly, I think people that serve on these review panels are aware of this challenge and are exploring different ways to try to manage it.

Let's go back to lab culture. We went from institute, so you obviously have been a wonderful mentor, and do you have any specific advice or observation with regard to mentorship and building an inclusive culture and how one may want to approach this, because I'm sure that's something that you probably within the institute, in your institute, but also in your lab, I'm sure you're faced with this question.

Well, I guess I think about it in a couple of ways. First of all, I feel that My job as a mentor is to help each person that I'm working with to do the very best work that they can do, and, and that involves understanding who they are as a scientist and a person, what their passions are, what they're good at doing, what they enjoy doing, the kinds of people they enjoy working with, you know, all of those things. And so, for example, when a new a new student or a new lab member joins my group, I try to spend enough time with them that I can learn about them a bit and find out how they like to do science and what they like to think about. And then to really help them get into a project and working with the right people where they can excel. And sometimes you get it right and sometimes you don't right away. And when I don't get it right, I try to figure out and really working with the person and asking, okay, So is the project not working well because the project is flawed in some way or we didn't have the right idea, or is it that something else is missing? You need a different type of expertise or you need more mentoring or something else. And so that's— and I've really come to this way of thinking about running a lab over time. I certainly didn't think about this for a long time when I had first started my lab. I was just trying to do science and I had projects that I was really excited about and I tried to get students excited about the things that I wanted to do. But over time, I think about it much more now with the perspective of, I guess it's partly aging at some level, but realizing that the most important thing that I can do now most likely is to help train the next generation of scientists, people who are going to be taking science into the future and trying to give them the very best preparation for the work that they will do in the future. It's also really fun and really rewarding, you know, when you get it right and you see how incredibly well people are doing that have trained in the laboratory. That's, that's the one of the greatest joys that I think we have.

Yeah, I agree, I agree. It's great when you find that fit for a person and a project.

Really great.

Yeah, so we have lots, tons of questions. I think we should move to the questions. Thank you so much so far, big picture, so there's lots of possibilities for questions. So let's go for the first one. What are the state-of-the-art challenges in near and far future applications of CRISPR technology? How is the CRISPR compared with other gene editing tools?

Well, I'll start with the second question first, and that is that, you know, what What makes CRISPR really powerful, frankly, is the mechanism by which it works, you know, the chemistry by which it works, because it uses a molecule of RNA, which is a chemical cousin of DNA, to recognize DNA sequences in a, you know, specific way. And so that mechanism is very easily manipulated by scientists. It's easily manipulated by bacteria too, which is why it evolved that way. That, you know, in the laboratory we can easily change this molecule of RNA to a desired sequence, and then that allows the very same CRISPR protein that uses this RNA guide to be used in any type of cell to, you know, manipulate any genome. And so that is just a very fundamental aspect of this technology that's distinct from earlier technologies for genome editing that relied on engineering proteins that had, you know, recognition capabilities for DNA, but— and which could work very well, but where you had to engineer a new protein for each experiment. It's just, you know, it's really different. So that's an important thing to appreciate about CRISPR. And beyond that, you know, one of the things that's really driven the field over the last decade is the fact that with that fundamental platform of, you know, mechanism of DNA recognition, you can do lots of creative things to DNA using that same recognition platform. And lots and lots of labs, including many in Boston and at the Whitehead, have been able to do this very beautifully, right? And where you can, you know, you can make chemical changes to DNA, you can make alterations to the, the chromatin state of DNA, so you can affect the levels of proteins that are made from specific genes without having to change the DNA sequence itself, something that Jonathan Weissman, again, his lab really pioneered. You know, I think these are things that make the technology very, you know, very useful and very versatile. What are the challenges? Well, you know, I think two that come to mind. One is delivery, how we deliver these editors into cells or tissues where they can be beneficial. And that's true whether we're talking about a human body or a plant or even a microorganism, right? You have to get it into the right cell where it can have a desired impact. And the other thing is ensuring precision of editing, because it's one thing to be able to, you know, hit a target, but with genome editing, what we really want to be able to do ultimately is to precisely dictate, you know, what change is made to the DNA. And that's still a challenge in the field, is ensuring that a precise change of our, you know, of our desire is made at a particular place in a genome. And so I think over the next, you know, certainly the next decade, but probably before that, frankly, those challenges will be addressed with lots of creative approaches that are already in progress.

So there are a couple of disease-focused questions. So how close are we in seeing real-world clinical treatments for cancer based on CRISPR?

Well, with cancer, I think there, I think it's pretty exciting to see the intersection between CRISPR and, you know, cancer immunotherapy, where it's possible to already, for certain types of cancer, to harness the immune system to go after cancer cells very effectively. For example, for diseases like melanoma, you know, this has been a very effective strategy. It's much harder with solid tumors, and I think over time, I hope that, you know, CRISPR will impact that field by making it possible to program immune cells more effectively to target different, different tumor types. So that's, you know, that's one area of cancer biology where I think CRISPR will have an impact. And maybe on the other end of things, more on the research side, I do think we'll continue to see advances with CRISPR as a discovery tool to understand better the genetics of cancer, the interactions between genes that lead to different types of cancer, and then of course, hopefully, how to treat them.

Now a more difficult one, I would say, is the possible use of the technology to study mental illness and potentially addressing mental illness with CRISPR. Yeah, it's a great question. Lots of questions.

Yeah, it's a great question, and I don't have an easy answer. I guess I would probably come back to the idea, at least at least for now, that one could imagine CRISPR being used to understand the genetics that lead to certain, you know, mental diseases. It's obviously difficult because you don't necessarily have good animal models. You can't really do this effectively in cells cultured in a dish for the most part. So, you know, it's hard, right? It's a very hard thing to study, but I do think that With all of the advances being made right now in neurobiology, even, you know, sort of understanding the different cell types that are in the brain and how they interact, I think that biology will intersect, I hope, with the kinds of genomics approaches that CRISPR enables, that you'll start to have a real genetic understanding of the wiring of the brain, and then, of course, how it may go awry in certain mental conditions.

Yeah, with all those prospects, there's a kind of a tricky question, and that is, what's your vision for equitable democratization of therapies that use CRISPR as their mode of operating to treat patients? And that's obviously— we hear about, you know, how many thousands and thousands of dollars these therapies would cost, right?

Yeah, or millions, right? Millions, yeah, right, yeah, right.

All these individualized focused, expensive.

Yeah, right. And thank you for that, for that question. It's a really important one, and it's something that I think about now daily. I mean, this was a big motivation for us starting the Innovative Genomics Institute a few years back, actually, is that it was clear that CRISPR has such incredibly exciting potential, and yet it'll never really reach that transformative potential on a broad scale unless we can make it affordable and accessible to, to any that can benefit from it ultimately. That's a tall order. It's a hard, you know, thing to contemplate, but I do think that in the case of CRISPR, at least, we can tackle it partly by just using that, applying that lens to projects that we work on at the Institute. So I'll give you a couple of examples. We just, we have an FDA-approved Phase 1 clinical trial that's underway currently for sickle cell disease. It's a 3-way partnership between the University of California, Berkeley, San Francisco, and UCLA. So the trial is actually being conducted by, by scientists at UCLA, but with technology that was developed at the Innovative Genomics Institute using CRISPR. And, you know, why run another sickle cell trial? There's several underway already. Well, we're the only nonprofit a nonprofit organization running a trial. And our motivation is that we want to do this in part to de-risk the technology to the point where we can partner with companies appropriately, but with some control over the cost of the therapy. And the other thing is that we have underway at the sort of in parallel with the trial where we have ongoing research that is focused on reducing the cost, for example, by coming up with new strategies for delivering the CRISPR editors into the bone marrow that won't require a bone marrow transplant for the patient, which would, you know, definitely reduce the cost and the, the need of hospitalization for each patient. So it's those types of things that I think one can apply to projects to, you know, really hopefully in the future drive down the cost per patient. Another example is that we, we have a clinical testing lab that we've set up at our institute that that right now is doing COVID testing, but in the future is going to be doing validation of CRISPR-edited tissues and samples from patients that we can control. And so it gives us a way to, in real time, be evaluating the efficacy of those editing experiments. And again, we hope to accelerate the testing of delivery strategies with new types of editors that will over time reduce the cost of this type of treatment.

Yeah, I think that's— because it's interesting, it's really the delivery and the efficacy more than almost the methodology itself, right?

I think at this stage, yeah, yeah, that's right.

So there are a few questions to the methods actually, which are really I think really interesting questions. How reversible are CRISPR edits? Once an edit enters an organism, can it ever be eliminated?

Well, yeah, it's a good question. I suppose one could edit it, do a second edit to remove it or change it furthermore. But probably realistically, I think that genome editing will be deployed in a variety of ways that will depend on the treatment that's needed. So for example, it's one thing if a patient has sickle cell disease and they need to have— essentially, they need their red cells, red blood cells to be replaced by cells that can make a form of hemoglobin, the protein that carries oxygen in the blood that's not prone to aggregation, which is what happens. And with sickle cell disease. So that's something that can certainly be done today using CRISPR technology. And, oh, I'm trying to remember where I was going with this. And this, oh, this was about the efficacy, right? Say again, Ruth? Reversibility. Oh, right, the reversibility, right. So in the case of a treatment like that, you know, you would really want that to be effectively a permanent change in the person. It might be very different if you have a gene that needs to be turned on, let's say, for a short period of time to provide an essential protein, let's say, during development that you don't want to have produced once the individual is more advanced, say, as an adult. And so there you could imagine using CRISPR differently, and I'm coming back back to the work of Jonathan Weissman at the Whitehead again to think about, you know, who's been working on ways of using CRISPR to not permanently alter the genome, but in fact to just make that kind of a transient change to the protein levels that might be coming from a particular gene. So I think we just have to think about CRISPR as, you know, a versatile tool that can be used in these different ways depending on the desired outcome.

Yeah, because also tissue-specific, obviously, right? We were talking about cancer cells, target to the cancer cells, so that would be probably another way. There's another sort of clarifying question: does the immune system fight CRISPR-modified cells?

Yeah, that's another, that's a really interesting question. So far it looks like not, and this is based on work that has been published from groups like Carl June, who's been looking at this for edited T cells and for various companies that are running clinical trials for genetic diseases. And so far, it really looks like those edited cells are not rejected by the immune system, although it's something that I think needs to continue to be investigated for each application.

And what are the biggest obstacles, leaving aside the ethical questions regarding the application of CRISPR in the treatment of genetic disease. We already talked about some of that.

Yeah, well, I think it's, you know, obviously, first of all, one has to know what gene or genes need editing, right? So that's, that's one challenge for sure. For, for many diseases where that isn't well understood, then I think that would be the first order of business, would be to figure out what are the genetics there.

The basics.

Yeah, to the basics, right? Yeah. Can then they— can they be manipulated? We didn't talk about accuracy of editing, but, you know, there's always the potential for what people call off-target editing. We know this can occur, and people still debate, you know, how worried should we be about off-target editing? And probably it comes down to, you know, what's the application, and what's the level of off-target editing, and is the off-target editing occurring in a place that might cause trouble? In the genome. And fortunately, there are lots of tools now for evaluating this. So the, you know, the scientific community has really focused on this question in a big way. So there's a large literature on the accuracy of CRISPR genome editing in different types of cells and under different conditions and with different types of editors. And, you know, I just think that again, it's the kind of thing where Obviously, the bar for clinical use has to be high. If you're— if we're doing research experiments or we're working in other systems, animals or plants, you know, the bar is probably lower. But in all cases, fortunately, we now have very good tools for evaluating the accuracy, and increasingly there are a wide range of CRISPR editors that have different intrinsic accuracies as well.

Thank you so much for answering all the questions. So there's the last question, which I think is a great one for a last question, and that is: looking back over your whole career, is there some question you ran across that seemed impossible to answer but now tempts you to take another crack at it?

Um, gosh, what a, what a wonderful question. I'm not very prepared to answer it, though. Well, you know, I just, I often think about, you know, I just, I, for me, I, it often comes back to the fundamentals, you know, about, about evolution and, and trying to understand something about why, you know, why does modern biology look and act the way it does? And currently, I guess the The area that I've gotten most intrigued about with respect to just very fundamental science is, you know, is kind of the microbial world. I have a wonderful colleague here at University of California, Berkeley, Jillian Banfield, who's also one of the directors here at our institute. And, you know, she's a real leader in the metagenomics of microbial populations, not only in places like the human gut, but she also works a lot in environmental samples. And the biology there is so incredibly interesting, and she keeps coming across things that are just, you know, really unexpected and unusual. She not only was one of the first labs to find CRISPR sequences in the first place way back when, but, you know, increasingly she's exploring the nature of inheritance in these organisms, and it turns out that it's not nearly as simple as as I certainly thought it was. And also the boundary between a living cell and a virus is a lot more fluid than I used to teach to my intro biology students here. So I think there's just— it just is a great reminder. I don't think I'm exactly answering your question, but I'm just telling you that this is an area of biology where I think there's a lot of interesting exploring left left to be done. We don't necessarily even know what questions to be asking. It's more about just kind of keeping your eyes open for interesting phenomena that then might alert you to a new, you know, a new activity in biology that would be exciting to explore.

Yeah, so who knows, right? Microbes and viruses have taught us so much of so much biology, right? And, you know, maybe the problem of how you go into tissues and how you deliver may also come from— Exactly.

—could be cracked by studying these organisms. I agree completely, yeah.

So Jennifer, thank you so much. This was so much fun, and we could be going on and on and on. Thanks for joining me today and making this definitely a memorable addition to our Director's Dialogue. I greatly enjoyed the conversation, and I'm sure our audience did as well. I'm sorry for those questions we were not able to answer. Thanks for joining us.