CRISPR-Cas9: Advancements and Ethical Considerations

Jennifer Doudna's work on CRISPR-Cas9 has led to groundbreaking advancements in genome editing, with potential applications in various fields.

### CRISPR-Cas9: From Bacterial Immunity to Genome Editing and Beyond
* Professor Jennifer Doudna's groundbreaking work on CRISPR-Cas9 began as **curiosity-driven research** on a bacterial immune system, which she later **repurposed for genome editing**.
* The CRISPR-Cas9 system allows for **precise DNA editing**, with potential applications in **research, agriculture, public health, and clinical medicine**.
* Somatic cell editing, like the ongoing trials for sickle cell disease, holds promise for **treating genetic disorders** without passing changes to future generations.
* 👶 The controversial topic of germline editing, as exemplified by the unethical "**CRISPR babies**" experiment in China, raises **significant ethical and safety concerns**.
* 🔬 Ongoing research is rapidly advancing the CRISPR toolbox, with innovations like **base editing**, **prime editing**, and **cell-specific delivery methods**.
* 🏥 Recent clinical trials have shown promise in treating genetic blood disorders and cancer using **CRISPR technology**.
* The future of CRISPR research will focus on **improving delivery methods**, **expanding editing capabilities**, and **addressing ethical and regulatory challenges**.
* 👩 Professor Doudna acknowledges the importance of **collaboration, funding, and the diverse team** at the Innovative Genomics Institute in advancing CRISPR research.

[Music] Please join me in welcoming to the distinctive voices podium, Barbara Meyer. In no time in human history have we ever faced more challenges and threats, and more opportunities and advantages. I'd like to argue that our speaker today, Professor Jennifer Doudna, offers us the wonderful opportunity of the advances that science can bring. It's a privilege and honor to be able to introduce my friend and colleague, Jennifer. In thinking about my introduction, I thought of two adjectives that describe Jennifer: genius and generous. I could add gorgeous to the G the triplet jeanne genius. Jennifer as a youth stomp the woods in Hawaii, dreamed drinked up nature, drank up nature, and realized how important nature was. She had a father who valued her ideas and opinions, and who encouraged her conversations with the dinner table, and that inspired her to go and go into deep challenges. That combination nature and dad. She went to Pomona is an undergraduate and excelled, of course. She went to Harvard med school and got her PhD with Jack Szostak, working on very, very innovative.

projects. Jack was thinking about the origins of life. Jennifer was thinking of RNA as an enzyme, and that was not really very thought of them. Jack won a Nobel Prize. Jennifer then went on to later went on to Tom Tech's lab. He want to know about price too. I think Jennifer has something to do with both of those and continue working on RNA as enzymes. She then she then went to Yale, and we finally, fortunately, were able to steal her to Berkeley. So what characterizes Jennifer, deep insight. Jennifer can look at a problem, see data, and see vision in that that no one can. And the perfect example of that is what she's going to talk about tonight. Chris Burke has nine. She saw the bacterial immune system. She realized its potential when no one really did. She figured out the mechanism for how this particular RNA directed protein could work. Not only said she was not satisfied with the mechanism, she wanted this approach. She realized this approach was extremely important for humankind, and she not only published

if she came to her colleagues, and she asked for collaborations, and she just did a wonderful job of promoting her enthusiasm for this project. The genius part is that there's more, she's won every award I've ever heard of, and more than I've never heard of. The generous part, Jennifer not only has a vision, she's generous in many ways. She's generous if she wants to share the science, she wants to make sure everyone has the science, but she's generous to society, she wants to make sure that that approach is properly deployed. Jennifer does not rest if things are done improperly, Jennifer does not rest if society does not use technical advantage as well, and so we welcome her here tonight to tell us about her approaches and her advances, the challenges, the threats, the opportunities of Christopher Cass 9. Thank You, Jennifer. [Applause] well, good evening, everyone, and I'd like to start by thanking the organizers, Theodore Barbara, and the National Academies for inviting me here, all of you for attending tonight.

Barbara, that was just an incredibly nice introduction. And what I would didn't mention is that I think you might have been the very first person that we spoke with after our initial work on CRISPR Castine about the opportunities to use it for genome editing in systems that Barbara's lab is studying. So forever grateful to you for that and your encouragement in that project. And what I wanted to do tonight is to share with all of you the kind of the journey that I've really been on over the last seven plus years working on a system that, as Barbara said, began as curiosity-driven science to understand a bacterial immune system called CRISPR. And our interest in this as a fundamental question in biology. And then share with you how that work morphed into a technology for genome editing and that we're where that's now headed into the future. It's a really exciting time in biology right now because we're kind of at a moment where we can think about genomes, the code of life, at extremes of scale on the

one hand, we can sequence entire genomes for less and less money and time. We're gonna have the hundred-dollar human genome at some point, no doubt. And we can also increasingly interrogate genomes and understand the information that they encode. And part of the way that we can do that is through the technology that I'll talk to you about tonight, CRISPR-Cas9. Now, we got into this not as genome engineers. In fact, that was not even on my mind when I was contacted by a colleague at Berkeley, Jillian Banfield. It was probably around 2005 or so, and she called me up one day and said, We don't know each other, but I'm working on something that I think you might find interesting. And so we met and we discussed a project that at the time nobody in the world was really working on. It was something completely new and very obscure, namely a suspected bacterial immune system that could allow bacteria to defend themselves against viruses. Now, Jillian's lab was not doing

experimental biology. They were doing computational work. They were sequencing bacterial genomes, and they found evidence of this bacterial immune system before anybody else had really caught on to it. Jill contacted me, thinking that our lab, as a biochemistry and structural biology lab, might have an interest in investigating how this system operated. So that led us to a fascinating series of experiments in which we began studying how these CRISPR systems actually function in bacteria. Now, this is a cartoon, a diagram. It's, oh, it's a, there's a lot going on here, but I'll try to explain this. So we're looking at a cartoon of a bacterial cell that is being infected by a couple of viruses. And you can see when these viruses infect, they actually inject their genetic material into the cell and start generating the products encoded here with the purpose of making more viruses. And that's a process that happens in essentially every cell type. All cells, probably, I think, are susceptible to viral infection. And in bacteria, if these bacteria have in their

genome. A CRISPR system is characterized by a set of sequences that allow capture of a little piece of viral DNA during the infection process. So, the cell can essentially grab this piece of DNA from the virus, store it in the bacterial genome. In the context of this CRISPR array, which is a very organized set of sequences that Banfield's lab originally identified, the storage of those sequences allows the cell to keep a genetic record of previous viral infection. The reason that Jill reached out to me about this, out of all the people that she could have contacted, is because our lab has had a long-standing interest in molecules called RNA that are made from DNA. They serve as an intermediary between the encoded information in the genome and the products of that information, which are primarily proteins and other RNA molecules. So, we started to investigate how these CRISPR sequences in genomes of bacteria get transcribed into RNA molecules that can be used to help the cell find and destroy these same viruses should they

try to infect the cell again. And so, this shows that after the cell makes an RNA copy of this CRISPR array with its stored sequences from viruses, those RNA molecules then combine with proteins that are known as CRISPR-associated, or Cas, proteins to form these RNA-guided proteins that surveil the cell, looking for matching sequences. Sequences that have a similar set of letters to what's found in the CRISPR RNA. If a match is found, then these RNA-guided proteins are able to capture that DNA and cut it up. So, it's a fantastic way that bacteria can essentially evolve in real time in response to viral infection and use the information from viruses against those very same viruses. So, there's a lot of really fun biology behind this. And I'm just gonna show you a video here that illustrates the way we imagine this process working in nature. So, here are viruses that are infecting a group of bacterial cells. The DNA gets injected into the cell, and if there's a CRISPR array in the genome, the cell can capture

a piece of viral DNA. Store it in this array, and then it's sort of marked by these repetitive sequences that flanked the pieces viral DNA that are stored, and then the cell is able to make a copy of that sequence in the form of an RNA molecule that gets processed into individual units that each contain a virally derived sequence, and then these RNAs combined with the second type of RNA called tracer that allows assembly with a protein called cast 9. And so these are n a guided proteins are then able to search the cell looking for a sequence of DNA that matches the sequence in this guiding RNA. And what you can see here is that when that match is found, the DNA unwinds, the protein cuts the DNA, and in bacteria, that leads to destruction of those cleaved molecules of DNA and protection of the cell from that virus. So a lot of fun biology there. Now as we were doing research on this project, initially I ended up going to a conference in Puerto Rico where I met another scientist, Emmanuelle Charpentier.

whose lab was working on a type of CRISPR system that at the time. My group, my lab, had not started to investigate. And it was a system found in a type of bacteria that infects humans. And Manuel's interest in this bacterium was as a, as an infectious agent in humans. And when we met at a conference, we decided that it would be very interesting to work together on the molecular basis for this new kind of CRISPR system in an infectious bacterium. And so, we started to work together on a protein called Cas9 to figure out how it functioned as an RNA-guided enzyme. And that led to a fabulous collaboration in which we figured out that Cas9 uses its RNA guide to interact with double-stranded DNA in the cell at a sequence matching the 20 letters of this piece of RNA. And again, as you, as I showed you on this previous slide, this would be in bacteria, would be an RNA that comes from that CRISPR array and a sequence originally coming from a virus.

and so when this interaction occurs cast 9 has the ability to cut the DNA strands. So it makes a double-stranded break in DNA. So DNA is a double helix. It's just like a piece of rope. This enzyme cuts both strands of that rope at a precise position that's marked by this guiding RNA. Now this research was being done by two of our lab members, Martin Yannick and my laboratory at Berkeley, and Christiansí, a grad student working in Emmanuelle's lab in Europe. And these two scientists working together over across the 6,000 miles or so separation figured out that this enzyme uses these 2 molecules of RNA for this kind of guided DNA cleavage activity. And they figured out a number of other aspects of this reaction that allowed us to make a form of the RNA that we called the single guide. So we could link together two separate molecules of RNA that are normally produced separately in bacterial cells, but we could produce them in the laboratory as a single molecule that we

called the single guide RNA that would have the information necessary for RNA-guided recognition of DNA. On this end of the RNA, and on the other end, a handle, a little structure required for interaction with Cas9 enzyme, and assembly into this functional complex. And it was really that key experiment that was done by Martin Jinek in my lab at Berkeley. When he did this experiment, showed the single guide could be used to program Cas9 to cleave DNA molecules of our choosing in the laboratory. That we realized that this project that had started as a curiosity-driven investigation of bacterial immunity was leading in a very interesting and exciting a new direction for us, which was the idea that gave us the idea that this could be used for genome editing. And to explain that, I want to show you a cartoon that illustrates what happens in plant and animal cells when those cells experience a double-stranded break to DNA. Because unlike in bacteria where double-stranded breaks pretty quick generate degraded DNA molecules, in plants and animal cells, and human cells, double-stranded breaks in the genome.

actually trigger DNA repair so these cells can recognize broken ends of DNA and fix them by recombination pathways. That trigger either a disruption to the DNA sequence at the site of the break or insertion of a new piece of DNA that can actually introduce new genetic information at a precise position. During this repair process, now we didn't figure that out. There were a wide range of scientists who had studied this process, including Dana Carroll who's here, and a number of others that are represented at this conference. And those scientists had recognized that a key to making targeted changes to genomes was to figure out how to introduce a double-stranded break at a desired position. And this is where Cas9 comes in because it turns out that bacteria had figured out how to make and program an enzyme to do exactly that, to program it to cut double-stranded DNA at a particular position. And to show you how this works as a genome editing tool, I want to show you this video, which shows back to a cell of a eukaryotic

organism with a nucleus, so a plant or animal cell. And here's this RNA-guided protein, Cas9, searching through the DNA of the cell. You can see it has a lot of DNA to search through, but it's able to do that quite efficiently to find sequences that match the sequence of the guide RNA. And now this is a bit of an artistic license here, but where you imagine that the enzyme forms this structure, we know that structure occurs because we can visualize it in the laboratory. And it leads to this kind of double-stranded break at a precise place in the genome that triggers DNA repair. And in this example, the repair introduces just a very small but targeted change to the DNA sequence. But as I'll show you in the latter part of this talk, there are now ways to use this system to introduce all sorts of different types of changes to genomes, but to do it in a targeted and programmable fashion. And so when we published this work in the summer of 2012, this was really the, in a way, the

dawn of this new era of programmable genome editing that we're now in the middle of because it gave scientists an opportunity to use this system to introduce changes to the DNA of any cell or organism that they might be investigating. And very quickly, it was clear that this bacterial system could be adapted readily to work in many different types of cells and in systems. And as we're hearing about at this scientific conference, there now a wide range of animals and plants and even human cells that have been edited precisely using this system. So what I wanted to do in the rest of the lecture tonight is to tell you a bit about what has happened over the last seven years since the origin of this technology, what is being used to do, why scientists are so excited about this, and then to say a bit about where this is going in the future. It truly is an exciting moment in biology right now. I think all of us that are working in this field have this sense that they're just

there's so many opportunities. There's so many exciting things that we can now do with the convergence of technologies. And it's really not just genome editing, but it's also the ability to sequence genomes readily. It's the ability to synthesize molecules of DNA very easily, to understand the organization of molecules inside of cells with new imaging technologies. All these abilities, capabilities, are converging to give scientists this incredible toolbox to start asking questions that just a few years ago none of us would have imagined that we would be able to do. So it really is an exciting time to be doing this kind of biology. So for all of you that are students in the audience, you know, you know this is really an exciting time to be entering the field of science. So let me tell you just a few things about CRISPR. And you know, I want to talk about opportunities and challenges. I want to just share with you very quickly some of the ways that CRISPR has really changed the way that we do science. And you know, I'm gonna for

the scientists in the audience, this won't be a surprise to you, but for some of the students, you know, I just want to give you a sense of the things that are now possible given this toolbox for genome editing. And I want to tell you a little bit of it. I'm going to give you examples in these four areas because it's really about not only fundamental research that can be done now that was very difficult to do or maybe even in some cases impossible to do in the past, but also opportunities in public health, in agriculture, and in biomedical science, especially in clinical opportunities with genome editing. And these are, you know, this is, you know, when I put together a talk like this, I have to say that there's so many examples that I could show you that it's, you know, you sort of feel like a kid in a candy store looking at the scientific literature around this, but I picked out a few examples that I think will you'll find really interesting. So first of all, in the research space, so one of the

things that's happened with CRISPR is that it's made it possible to do genetics on organisms that in the past would have not been available for genetic analysis by scientists. And this is a great example of this. So this is a was a wonderful story that came out last summer about CRISPR baby snails. And this is, you know, there's been a question in developmental biology for a long time about organisms that have natural handedness to their body design. And this is evidence in sales where typically these snails have a left-handed, sorry, I have a right-handed twist to their shell. You can see that they're very rare to find shells nails shells with the opposite opposite handedness in nature. And scientists have wondered for a long time why is that what are the genetics of that and using CRISPR it was actually possible to interrogate the genome of the snail to find it turns out a single gene responsible for that handedness. And so this article that was published earlier in 2019 showed that

you could, in fact, manipulate these snails using CRISPR to change the handedness of their shells, without affecting other properties of these animals. By doing this kind of very targeted genome manipulation, something that in the past would have been very hard to imagine being able to do. A second example that I want to share with you is in the area of public health, and this is a topic that we're actively discussing here at this conference with the work of Andrea Crisanti and others who are working actively on using genome editing to introduce changes to organisms in a fashion that allows the spread of a trait by a non-Mendelian type of process. This is a cartoon that I took from Science News that illustrates the way that what we call a gene drive actually works. So, a gene drive is simply a way to introduce a trait through animals in a population or organisms in a population very quickly in a way that doesn't require normal inheritance mechanism. So, over here you can see that in this population of

mosquitoes. If we have a trait that's being passed down through normal Mendelian types of processes, then you can see that this trait is inherited according to this cartoon and is happening in sort of a linear fashion. There's no spread of this trait across organisms horizontally in the population, but if we hook that trait up to a genome editor that's able to very quickly insert itself into genomes that it comes into contact with, then we can have a situation like this where animals that inherit this trait along with the gene Drive are able to give it to animals that are in this have this horizontal relationship, and very quickly the trait can spread through the population. Now why might that be interesting beyond just sort of a curiosity in science? Well, scientists for a long time before CRISPR came along were imagining that if you could do this kind of manipulation in organisms like mosquitoes, you would have a very powerful tool for controlling these populations and maybe either reducing them or making them incapable of spreading of a parasite or an infectious.

disease. And so, that has now become a reality with CRISPR, because it's now possible to do this kind of gene drive reaction in laboratory settings with animals like mosquitoes and fruit flies. And so, that raises a very interesting conundrum of asking, on the one hand, there could be a very profound public health benefit to this kind of manipulation, but on the other hand, there could be environmental impacts that are either unintended or were difficult to become difficult to control. And so, that's another, this is an area of discussion that we are as a scientific community now faced with, given the reality of a technology that allows this kind of effective gene drive in populations like mosquitoes. A third example I want to share with you is in agriculture. In this, I've showed this slide before, but I really love this. This is the work of Zach Lippman, who's here at the conference, who showed that you could use CRISPR to manipulate genes in tomatoes that control the production of fruits and allow manipulation of the

yield of tomatoes in these plants. And he's got a gorgeous slide that he showed at a recent Cold Spring Harbor meeting where he's able to actually alter the fruit yiii in tomatoes in a very precise fashion with a wide range going from none to huge numbers of tomatoes by manipulating the genetics of these plants in a way that requires the targeted approach targeting a particular gene and its production in these organisms in these plants. Now, this is a really cool use of CRISPR, but you can start if you start thinking about it, and it turns out that the genetics in tomatoes and that allow this kind of manipulation occur in other plants as well. So you can imagine ways to manipulate crop yields using this technology in ways that could be incredibly important in different environments around the world. And so something that started as a curiosity quickly becomes a tool that could have incredibly important practical applications in food production. And then finally, I wanted to just point out that in the biomedical space, and I want to

want to I'll talk now quite a bit about opportunities in clinical medicine. But I want to point out that the CRISPR casts enzymes are interesting not only for genome editing in biomedical use, but also for diagnostics. And this is an example that just shows that you can actually use these RNA-guided proteins. CRISPR casts enzymes. This is a protein called cast 12 that has a similar RNA-guided DNA cutting activity, like I showed you for Cas9, that can be used as a detector for interaction with specific DNA sequences that leads to a release of a fluorophore that can be detected very easily in a laboratory or clinical setting. And so this is a way that scientists are now exploring how we can use these bacterial proteins and take advantage of their RNA-guided activities for detection of specific sequences that could allow detection of viral or bacterial sequences to identify infection, as well as potentially to look for sequences of DNA that correspond to tumor development and help scientists with diagnostic applications.

that might have might otherwise be very difficult to to develop. So I want to now turn to to thinking about biomedical applications. And if you read the the popular media about CRISPR, this is by far the the subject that gets the probably the bulk of the attention because there's a lot of fascination with thinking about being able to manipulate our own DNA for the purposes of either mitigating disease or even potentially curing genetic disease or introducing traits into the human genome that might be in some way desirable. Now to to explain this, I first want to point out that we can think about genome editing in two different types of cells. We can do this in somatic cells, which means making changes that are not heritable. They don't get passed on to future generations. And so only a single individual is affected. And that contrasts with what happens when genetic manipulations are made in germ cells. So these would be eggs or sperm or embryos where the changes become heritable and they affect not only an individual but their offspring as well. And you can

immediately think about this. See that there's a really important and profound distinction between these types of genome editing. Now, I think that the vast majority of biomedical applications, at least in the near term, for genome editing, are going to be in somatic cells. There'll be changes that affect individuals, and I want to share with you one example of this that I think is likely to be coming down the pike relatively quickly for curing a disease that has been well known for a long time in human populations, and the genetic cause has been defined for decades, and yet we haven't had any way of dealing with it, certainly not at its core cause, and that's sickle-cell disease. So, this is a slide that just illustrates the mutation that gives rise to sickle cell anemia. It's a single change in the DNA of the human genome. Imagine that it's a single base pair in a single gene for beta globin that leads to an altered protein sequence. That means that the

cell instead of making a normal protein that's required for carrying oxygen in the blood. A mutant protein is produced with this altered amino acid due to the change at the DNA level. That has a very profound impact on patients because it means that the resulting cells, instead of being nice round blood cells like this, they can easily pass through tiny blood vessels and capillaries. Form these sick old cells that have a tendency to occlude capillaries and lead to all kinds of problems in patients requiring blood transfusions and cycles of normal life and then crises where these patients have to be in the hospital. And not too long ago, a film crew came through Berkeley and they explained that they were doing a film and making a documentary about genome editing. And they decided to focus on sickle cell disease as a really interesting example and sort of a thread that links together a number of disparate discoveries in science that are converging to give rise to a future that may include an actual cure for sickle cell anemia. And I want

to show you a clip from this film that begins with a patient that suffers from sickle cell disease. David, who goes to a lab at Stanford to learn about CRISPR. So, let's take a look. So now we're mixing the cells with the CRISPR. Once it's into the cell, that starts the editing process. We can't see that, we just know it happens. I don't know how, out of all the genes that you have, that it targets the one that's doing sickle cell and not the thing that's making you grow hair. Oh, but it does, apparently. That's cool. And so, this film, this clip, is every time I see it, is so meaningful, it's so profound for me because we can see the potential of this technology. And to see this, this film, and to see David going to Stanford and working with Matt Porteous, whose lab is actively working on a gene therapy that will take advantage of CRISPR Cas9 for treating sickle cell disease and seeing his own cells being

edited with this technology, and realizing that this cure may be on the horizon for him is just amazing. And to think that this is happening in just a few years with this technology is truly extraordinary. So this is the film credit, encourage you to see it when it comes out. And for applications in somatic cells, I think that many of us are really of the opinion that we're really on the verge of some exciting opportunities, and potentially even cures, genetic cures for diseases like sickle cell anemia and others where there's a well-defined single gene that has a mutation that could potentially be corrected or mitigated using CRISPR-Cas9. But now I want to turn to talking a little bit about heritable gene editing. So this means germline editing, making changes in an animal or any organism really that lead to changes that can be passed on to future generations. So this was an application of CRISPR-Cas9 that happened very early on.

showing you a picture of using germline editing in piglets, but in fact, it was an experiment done by Rudolph Vanishes lab at MIT in mice. That showed first that you could use CRISPR cassadine for germline editing in mice. And that technology took off very quickly, allowing scientists to make animal models, mouse models of human disease quite readily using germline editing with CRISPR cast 9. And so for me, you know, with my biochemistry add-on, you know, when we had first published its work, and then very quickly, it was clear that this technology was useful in the germline of an animal like a mouse. It became obvious that it would be very likely to work in other types of germ lines as well. And I started to imagine that, you know, it might be possible to do this in the human germline. And so I, you know, I was, I was very, very uncomfortable with this thought initially, and it seemed just sort of fantastical to imagine.

manipulating human DNA in embryos in ways that could allow scientists to change fundamentally human genetics, and make changes that would become part of the whole person, and alter who they were, and alter who their kids were in the future. And I started thinking about this, and you know, I think my initial reaction was to try to run the other way, and I thought that might be nice if bioethicists and people that think about these sorts of questions professionally would take up this issue. But in discussions with my colleagues at Berkeley, in particular, I realized that scientists who are involved in technologies can't run the other way. They really have to be embracing what's happening, not necessarily liking it, but owning it, and getting involved in the very important conversations that need to happen as technologies are unfolding. And so, that led me to organize with my colleagues at the Univision omics Institute, a number of whom are here at this conference. Jonathan Weissman, for example, we organized a conference in early 2015.

discuss human germline editing, and that meeting had just a small group of people. But importantly, it included two scientists, David Baltimore and Paul Berg, who had been involved in discussions around the ethics of molecular cloning in the 1970s and how scientists had grappled with that technology at the time. And its potential dangers. And that meeting in that we held it up in the Napa Valley to this publication in which this group of people that were at the conference wrote an article that was published in Science magazine proposing what we called a prudent path forward with CRISPR-Cas9, especially for thinking about applications in humans and applications in the human germline. And so what happened next was very interesting because she owed her earn of and his colleagues published a paper at right around the same time with a very similar sort of set of concerns about human germline editing. And then the National Academies of Science got involved and the Royal Society in the UK. And this has led to now two

international meetings on this topic and a report that was produced by the National Academies in the spring of 2017 on human genome editing, and especially human germline editing, and how this should be managed. This was an effort by the scientific community globally really to grapple with this challenging question: what do we do now that we have a powerful tool that allows us to manipulate the DNA, even in human embryos, in ways that could fundamentally alter human genetics or even human populations if you start to imagine this being widely deployed? And I think many of us hoped that this would set guidelines that would be respected by scientists globally, who would agree with us that it was not a good idea to apply a technology like this in the human germline, certainly before the technology was truly vetted and before we understood well what manipulating human embryos might do. And then you can probably imagine my combination of surprise, shock, not complete surprise, maybe due to, you know, knowing the types of people that we're thinking about.

CRISPR-Cas9. I received an email in November of 2018 from this gentleman, Dr. He Jiankui, announcing that he had used CRISPR-Cas9 in twin baby girls in China to make changes to their DNA that would, in principle, protect them from HIV infection. Something that sounded good, but sort of a noble purpose. But when the details were announced at the meeting on human genome editing that was held in Hong Kong that year, and this is a picture of him presenting at that conference, it was clear that everything about this study was deeply flawed. And you know, we just had the one-year anniversary of this, and I've had, you know, many reporters have been asking for comments about this, and you've probably seen articles about it. And I think in the year that's passed since this announcement, many of us have had an opportunity to reflect on what was done and you know, think about, you know, how we might have avoided this situation and what we do going forward to avoid misuse of this technology in the future.

there are no easy answers, that's for sure, but I did want to show share with you one interesting scientific detail about this study that was revealed by Dr. Ha in this presentation that he gave in Hong Kong. That shows scientifically or technically why this was a really an appropriate thing to do. And so this is a cartoon that was put together by Shaun Ryder, who's a professor at University of Massachusetts. And what Shaun did was to simply take the data that Hood John Cui presented in his own slides and put it together as a comparison. And you don't even have to see the details here to read just want you to notice that the top two lines don't look like the bottom of three lines right because the top two lines represent on the top the natural sequence of a gene known as CCR5 that is responsible for, in it, encodes a protein required for HIV infection in humans. And then below it is a drawing that illustrates a natural mutation of a deletion of 32 base pairs that occurs very rarely in the human.

population, but gives those people protection against HIV infection. And what Dr. Ho was attempting to do was to introduce this change into the germ line of these human embryos. But when he sequenced their DNA, what he found was that although changes were made to this CCR5 gene, the change, the details of those changes are different than what you can see here. None of these changes look like this one. And that means that the changes introduced in these baby girls were changes that, to our knowledge, have never been seen in the human population and have never even been tested in animals. Sort of a horrifying thing. And then, of course, the ethics around consenting the parents for these children was deeply flawed, etc. So, I think for many of us, this was really a, you know, sort of a really a horrifying moment of recognizing that more needs to be done to try to control the use of this technology and make sure that it's used responsibly in the future. So, are we on the verge of CRISPR babies?

and and more more applications of this type. I certainly hope not, and I do want to point out that although these all of these various applications might look intriguing, for the most part, these are all characteristics of humans that have many many genes that contribute to them, and for the most part, we don't know what that constellation of genetics actually looks like right now, so we're sort of protected by our own igner, I suppose, but I think it's very critical that the scientific community and regulatory agencies work together to find a framework that will create a much stronger set of protections from irresponsible uses of CRISPR cast nine, like like CRISPR babies. And unfortunately, the World Health Organization and the National Academies have both convened International Commission's that are working on this very actively, and again, there are people at this meeting that are participating in this effort, and our hope is that these recommendations that come out of these the Commission work that's going on will provide a foundation for future regulations that can be put in place by.

appropriate agencies and governments. So, that being said, genome editing is speeding forward at an incredibly rapid pace. And just in the last couple of minutes, I want to share with you some things that are happening that I think are really exciting and will show you what's the what I think is coming down the pike in the very near future with respect to manipulating genomes. So, first of all, these are really the key areas that I think are going to lead to, you know, tomorrow's breakthroughs. One is advancing the tools for genome manipulation, and the second is figuring out how to deliver gene editors into cells in a specific way, and thirdly is figuring out how to make sure they work and they work correctly, and also to figure out how to manage the ethics around certain types of applications, in particular like germline editing. And just to give you a sense of the pace of development in the field, it's truly astonishing how rapidly now this whole CRISPR-based toolbox is evolving in real-time in laboratories.

only is it possible to make insertions deletions to control transcription that means controlling the output of genes and cells to control what are called epigenetic changes which again can affect the output of genes and the production of certain proteins and cells and to do that in a targeted fashion but it's also increasingly possible to do things called they're shown here one of them is base editing meaning making chemical changes to a single nucleotide in DNA without having to trigger a cut first using caste 9 in a mode that allows that kind of targeted based editing to genomes. Secondly to make changes that allow a scientist to target a region of a genome and then mutagenize it making lots of different variants of a gene in one just in one place in the genome and then test the effects of those mutations in the laboratory and then thirdly something very new that's been published called prime editing that allows introducing what are called snips this stands for single nucleotide polymorphisms making very small but

targeted rewrites to a specific section of a genome. And these are all tools that are coming along based on this RNA-guided fundamental activity of this bacterial enzyme CRISPR Castine. This is a little set of cartoons. And if you're interested in this, especially for the students, I to the innovative genomics dot org website that has all of this information in more detail. But I wanted to just ill in every case, these are versions of the caste 9 protein that allow different types of manipulations to be made to genomes. And if you look at the dates here, you can see that you know what started off in June of 2012 with the publication by Emmanuelle Charpentier and our lab has now evolved at these, you know, sort of over the last seven years in all these different ways of manipulating genomes in a precise program fashion. Now I was at a conference in Cold Spring Harbor just a couple of months ago where some of this, the latest and greatest advances in this technology.

were being developed, and I really literally had chills going down my spine because you could see in real-time how fast this technology, this whole toolbox, is developing. And it's amazing that these days when a paper is published in a journal, scientists around the world will read the paper, and within weeks they're taking that new iteration of the tool and applying it in different cell types, making it better, seeing what works, what doesn't work, and then for things that are imperfect, working to fix them and make them better, whether it's more accurate or more efficient, or what have you. And so it's truly astounding. I really think we're within about five years of having a toolbox that will allow scientists to make essentially any change with absolute precision in any genome. I think we're really just a few years from that, and we already have lots of the ability to do this in the laboratory. And so then the big question, you know, in thinking about applications of those tools, is how do we get these genome editors into cells.

where they're needed, and so there's a lot of interest in cell type-specific editing, making targeted genome edits in cell populations and in tissue, so not just doing it in cultured cells in the laboratory, but being able to actually introduce these into a whole organism and get editing where you want it. Innovation is going to be critical for this, and so increasingly at the Innovative Genomics Institute at Berkeley and UCSF, we're very keen to figure out how to solve this delivery problem, and I think there will be many solutions to it, not one solution, but I think it's a really important bottleneck that we're facing currently that needs to be addressed for genome editing to have maximum impact in the future. Two very quick science updates. So this is just work from our own group, so one of the things that we've been working on with Matt Francis in chemistry at Berkeley using an enzyme called tyrosinase that allows linking together of two different proteins using natural amino acids.

are exposed on the surface of those proteins. And this is an example where we use this type of chemistry to link together CRISPR caste 9, the gene editing molecule, with very small proteins that allow cell penetration. And when we do that, and you don't have to really read the labels here, but I'll just point out that this is an experiment where we're looking at genome edits in cells using different iterations of CRISPR caste 9. And we do this with CRISPR caste 9 that's been linked to cell-penetrating peptides. We get very efficient editing without having to do any kind of other manipulation to the cells. We just add this cell-penetrating form of caste 9 to the cells, and it naturally goes in and changes the DNA. Another idea that we have that we're actively working on and very excited about is using virus capsids to deliver CRISPR caste 9 to specific types of cells. And so this just takes advantage of what viruses naturally do. They have an ability to get

into certain types of cells, and we can use that. We can gut the viral capsid of all of the genes that make a virus, so it's not and no longer an infectious agent, but instead encapsulate the gene editors, but allows them to get into certain types of cells. And this is a recent experiment that we did. We have colleagues at UCSF, Alex Marcin in particular, where we took a mixed population of immune cells that are marked by these cell surface receptors, either CD8 positive cells or CD4 positive cells. They're all together in a mixture, and when we have these virus-like particles with a cell surface viral surface molecule that allows recognition of the CD4 positive cells, we start to see editing of just those cells in this mixed population. And we've got some much more recent data that shows much higher efficiencies of editing, so we're really excited about this. We think this ultimately could allow editing of immune cells in a patient that would avoid having to do a bone marrow transplant, which is what has to be done today even.

for sickle cell disease this is being you know sort of very actively developed in companies. Some recent announcements that were very exciting to this whole field showed that CRISPR casts nine can be used to treat blood disorders. This was very small just two patients, one with sickle-cell disease, the other with a disease called beta thalassemia, but in both cases it looks like CRISPR cast nine was not only safe but it was also effective at treating the underlying genetic cause of those diseases, giving the whole field the sense that were on the verge of applications like this so that will have real impact for patients. And then this was an announcement from the University of Pennsylvania Karl Joon's group showing that editing immune cells for cancer patients at least was a safe application of CRISPR. Not clear yet if it's efficacious if it's working or not but at least it didn't have any toxic effects in these types of cells so the power of RNA guided gene regular gene regulation is going to continue.

this you know the whole toolbox built around CRISPR casts proteins will continue to expand. I think delivery and control of these reactions are key. And by control, not only chemical control, but also you know societal, and then sort of regulatory control. I think are going to be really key in the future. And fundamental research continues, and you know we're, we're, we're having a lot of fun working with Jill Banfield's lab, our original collaborator on CRISPR, to look for not only new CRISPR systems, but maybe what's, what's, what's next, what's beyond CRISPR. And without, I'd like to just acknowledge a fantastic team. So, you know, running a research lab at Berkeley is, is amazing because I get students from all over the world that come together to work on scientific problems. And occasionally we even go out of the lab and go to baseball games, which is what you're seeing here. And huge thanks to our, our collaborators, our funding agencies, and of course to the innovative genomics.

Institute, which is a tri-institutional partnership, makes working in the Bay Area and working on genome editing in the Bay Area a really amazing experience. Thank you.

Genome Editing with CRISPR-Cas Systems: Challenges and Opportunities

Distinctive Voices

The Journey of CRISPR-Cas9: From Curiosity-Driven Science to Genome Editing

Professor Jennifer Doudna's work on CRISPR-Cas9 began as a study of a bacterial immune system before evolving into a revolutionary genome editing technology.

Her research has not only advanced our understanding of biology but has also opened up new possibilities for genome engineering.

Doudna's journey started with curiosity-driven science, led by a colleague's discovery of a bacterial immune system, and progressed into the development of CRISPR-Cas9 as a powerful tool for genome editing.

The Role of CRISPR in Bacterial Immunity and Genome Editing

A CRISPR system allows bacteria to store viral DNA sequences and use them to fight off future infections.

The collaboration between two labs led to the discovery of how the Cas9 protein functions as an RNA-guided enzyme to cut DNA strands.

This research also revealed the potential for CRISPR to be used in genome editing in plant and animal cells.

The Revolutionary Impact of CRISPR Technology

CRISPR technology triggers DNA repair and allows for the introduction of new genetic information.

It has revolutionized genome editing, providing scientists with the ability to make targeted changes to DNA in a wide range of organisms.

The technology has opened up new possibilities for fundamental research, public health, agriculture, and biomedical science, with the potential for significant advancements in each area.

The Diverse Applications of CRISPR Technology

CRISPR can change the handedness of snail shells and is used for targeted genome manipulation.

The technology is also being actively explored for public health purposes, such as controlling mosquito populations and the spread of infectious diseases.

In agriculture, CRISPR is being used to manipulate the genes of plants, potentially revolutionizing food production.

Additionally, CRISPR's RNA-guided proteins are being investigated for diagnostic purposes in biomedical applications, offering potential for disease detection and genetic analysis.

The technology's ability to edit both somatic and germ cells raises ethical considerations in terms of heritability and long-term effects.

The Future of Genome Editing: Somatic Cells vs.

Genome editing is expected to have a significant impact on curing diseases like sickle-cell anemia in somatic cells in the near future.

The use of CRISPR Cas9 for germline editing has shown potential in creating animal models of human disease, but the ethical implications of applying this technology to the human germline are a major concern.

The growing role of scientists in the ethical discussions and the organization of a conference to address these important issues highlight the need for careful consideration of the implications of genome editing.

The Ethical Challenges of Human Germline Editing

A small group of scientists, including David Baltimore and Paul Berg, proposed a cautious approach to CRISPR-Cas9 for human germline editing in a publication in Science magazine.

He Jiankui's controversial use of CRISPR-Cas9 on twin baby girls in China highlighted the lack of responsible oversight and ethical concerns.

The scientific community, along with the World Health Organization and the National Academies, is now actively working on establishing regulations to prevent the irresponsible use of CRISPR-Cas9 in human germline editing.

Genome editing is rapidly progressing, with a focus on advancing manipulation tools, delivering gene editors into cells, and addressing ethical considerations, especially in germline editing.

The CRISPR-based toolbox is evolving in real-time, allowing for targeted gene editing, epigenetic changes, base editing, mutagenesis, and prime editing.

The development of these tools is happening at a staggering pace, with the potential to make precise changes to any genome in the near future.

Efforts are also being made to solve the delivery problem of genome editors into cells, which is a critical bottleneck for maximizing the impact of genome editing.

Researchers are using cell-penetrating peptides to efficiently edit the genome without manipulating the cells, and they are also exploring the use of virus capsids to deliver CRISPR-Cas9 to specific cell types.

CRISPR-Cas9 has shown promising results in treating blood disorders and editing immune cells for cancer patients, demonstrating its potential for real impact in medical applications.

The expansion of the CRISPR-Cas9 toolbox and the importance of delivery and control of the gene editing reactions are key areas of focus for future research.