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Constructing living cells
Scientists across the world are conducting research into cells. But, despite all of their efforts, cells still harbour many secrets. However mind-blowingly complex cells may be, though, increasing numbers of scientists are starting to believe that it should be possible to construct a cell from separate building blocks, one step at a time. TU Delft has the knowledge to play a pioneering role in this process. On one of the walls of Marileen Dogterom's office, head of the department of Bionanoscience in the Faculty of Applied Sciences, there is an enormous drawing of the interior of a cell, with swirling proteins in all shapes, colours and sizes. A long string of DNA winds its way through the clumps. It is quite clear what the primary area of focus is here: the cell. But the research conducted in the department is about more than learning to understand how a cell works. The TU Delft scientists want to go a step further: they want to construct a synthetic cell, together with their counterparts in the Netherlands and abroad. The relatively young field of research that designs new biological systems using existing building blocks is referred to as ‘synthetic biology’. It is a specialist field that is clearly on the rise. Borrowing principles Marileen Dogterom herself primarily conducts research into the physical processes within cells. She addresses such questions as: ‘What forces play a role in processes within a cell?’ and ‘What is it that gives a cell its shape?’. A key area of Dogterom's expertise is microtubules. These are hollow protein tubes that form part of the framework of the cell, the so-called cytoskeleton. ‘We do not plan to precisely mimic any existing cell type,’ says the head of the department about the ambitious plan to create a synthetic cell. ‘Instead, we intend to borrow principles from simple systems such as yeasts or bacteria and then link them together.’ Borrowing principles and linking them together may sound simple, but there is still a lot to research and to learn, even about rudimentary biological systems. Research conducted by Craig Venter, the molecular biologist who became famous for being the first to sequence the human genome, confirms this. In 2010, Venter created what he described as a ‘synthetic life form’ by removing the contents of a bacterial cell, replacing it with DNA developed in the lab and bringing it back to life. It proved to be considerably more difficult than he thought it would be. Minimal cell In 2016, the same Craig Venter also presented a minimal cell. ‘Venter's group had taken a simple bacterium with a genome of around 1,000 genes and started taking bits out of it’, explains Dogterom. ‘After removing a piece of DNA, they would check to see if the cell was still functioning.’ By gradually shaving off the genetic material gene by gene, the group eventually discovered a minimum number of genes that the bacterium needed to keep itself alive. That magic number? 473 genes. Fewer than that and the cell no longer functioned. Craig Venter's ‘minimal bacterium’ is interesting, but also slightly surprising. Because if you look at the separate components that experts believe are needed in order to construct a complete cell from the bottom up, and the genes that code for them, 473 seems like a very large amount. Coordination and timing Christophe Danelon , who also conducts research into synthetic cells with his group in Delft, says he is aiming for a ‘global approach’. Danelon: ‘We do not focus on a single component, but we attempt to construct the different modules we need simultaneously.’ Not so long ago, for example, Danelon's group succeeded in artificially synthesising phospholipids. These fatty chemicals form the ‘vesicles’ in which the other parts of the synthetic cell will soon have to do their work. Danelon estimates that the components needed for a minimal cell can be made using approximately 150 genes, which is not even a third of Craig Venter's 473 genes. This therefore raises a question: will these 150 genes work together when they are combined into one singe strand of DNA? Danelon suspects that the genes in Venter's cell whose purpose is still unknown may be involved in such things as the coordination and timing of cellular processes. Ultimately, everything has to happen in the right sequence. For example, it makes no sense for a cell to start dividing if no copy has yet been made of the genetic material. Cutting through the middle Cees Dekker is the third leading TU Delft researcher involved in the project. Just like Marileen Dogterom, he has a strong physics background. The two scientists also share a refusal to be deterred by the complexity of the cell. ‘It is absolutely true that life is enormously complex’, says Dekker. ‘But does that mean we should say: it is so complicated, let's not even start? Or should we say instead: We see an opportunity to construct test systems and look at what we understand about them? The latter, in my view. I am a scientist because I want to understand how nature works.’ Dekker's group is focused on synthetic cell division, among other things. Some time ago, his group contributed to a study that demonstrated how the protein ring that cuts through the middle of a cell during cell division is constructed. Dekker: ‘I also conduct research into other biophysical processes, such as the spatial structure of DNA and proteins in cells. For example, we manipulated the shape of E. coli cells to see how DNA and proteins organise themselves.’ As well as providing some important scientific insights, this research resulted in the following extraordinary image: living bacteria in the form of the letters TUDELFT. Black box The Delft researchers emphasise that building a synthetic cell is above all a fundamental challenge. It is all about curiosity, about discovering how the cell works. But they are also convinced that the synthetic cell will result in all kinds of practical applications. ‘Just look at microbiology’, says Dekker. ‘In the beginning, research in that field was also driven by curiosity. But since then, a huge industry has emerged that uses bacteria as little factories to make all kinds of different substances.’ Marileen Dogterom expects that research into synthetic cells will result in medical breakthroughs: ‘When you administer a drug to someone, you are doing something with the molecules in their cells. Hopefully, that will have a favourable effect on the disease you are trying to combat. But in fact, you are putting those drugs into a kind of black box, because you do not know exactly what it is they do. This is why side effects are difficult to predict.’ The idea is that a better understanding of how cells work will enable us to intervene more effectively in the processes that make us ill. Ethical discussions Alongside practical applications, the construction of a synthetic cell is also likely to provide us with a greater understanding of what ‘life’ is, exactly, and how it began. ‘We have been doing cellular research for 70 years, and we have made tremendous progress’, says Dekker. ‘But no one knows how, four billion years ago, life first formed from separate biological components such as amino acids and lipids. That is a fascinating scientific question, and building a synthetic cell can help us find the answer.’ It may also raise ethical discussions on the issue of whether, as a scientist, you are permitted to ‘play God’: in other words, whether you should want to create life at all. ‘But’, says Dekker, himself a man of faith, ‘there is a perfectly good answer to that question. Moreover, it is doubtful whether the wider public will have such strong concerns about a primitive form of life. Currently, it is mainly journalists who raise the issue.’ Nevertheless, philosopher Hub Zwart from the Radboud University has now joined the Dutch consortium that has been awarded a prestigious NWO Gravitation grant for research into the synthetic cell. Linking pieces together How long will it be before the synthetic cell is a reality? It is difficult to say. ‘I am optimistic that, on a ten-year time scale, we will be able to make use of minimal components to make a system that can divide autonomously’, says Cees Dekker. Marileen Dogterom is slightly more tentative: ‘I am certain that it is possible to build a synthetic cell, but I am unsure whether that will be achieved within ten years. We are already capable of getting the pieces up and running. The great challenge is how we link these pieces together.’ Christophe Danelon loves his work and is quite happy for the research to take a long time. ‘I think I may have to sabotage the experiments when I sense that we are on the verge of a synthetic cell’, he says jokingly. But even if it proves possible to build a functioning, self-dividing cell, the question is whether that will conclude the project, says Dogterom. ‘Ultimately, it is about how you define success. For example, should a cell also be able to adapt, to evolve? What would you need to add to a synthetic cell to make that possible? And do we not also want to understand the interactions between cells?’ To ask the question is to answer it. And so, Christophe Danelon does not have to worry. There is more than enough work to do. Marileen Dogterom +31 (0)15 27 85937 M.Dogterom@tudelft.nl Cees Dekker +31 15 2786094 c.dekker@tudelft.nl group website Christophe Danelon +31 (0)15 27 88085 c.j.a.danelon@tudelft.nl This is a story from Applied Sciences On one of the walls of Marileen Dogterom's office, head of the department of Bionanoscience in the Faculty of Applied Sciences, there is an enormous drawing of the interior of a cell, with swirling proteins in all shapes, colours and sizes. A long string of DNA winds its way through the clumps. It is quite clear what the primary area of focus is here: the cell. But the research conducted in the department is about more than learning to understand how a cell works. The TU Delft scientists want to go a step further: they want to construct a synthetic cell, together with their counterparts in the Netherlands and abroad. The relatively young field of research that designs new biological systems using existing building blocks is referred to as ‘synthetic biology’. It is a specialist field that is clearly on the rise. Borrowing principles Marileen Dogterom herself primarily conducts research into the physical processes within cells. She addresses such questions as: ‘What forces play a role in processes within a cell?’ and ‘What is it that gives a cell its shape?’. A key area of Dogterom's expertise is microtubules. These are hollow protein tubes that form part of the framework of the cell, the so-called cytoskeleton. ‘We do not plan to precisely mimic any existing cell type,’ says the head of the department about the ambitious plan to create a synthetic cell. ‘Instead, we intend to borrow principles from simple systems such as yeasts or bacteria and then link them together.’ Borrowing principles and linking them together may sound simple, but there is still a lot to research and to learn, even about rudimentary biological systems. Research conducted by Craig Venter, the molecular biologist who became famous for being the first to sequence the human genome, confirms this. In 2010, Venter created what he described as a ‘synthetic life form’ by removing the contents of a bacterial cell, replacing it with DNA developed in the lab and bringing it back to life. It proved to be considerably more difficult than he thought it would be. Minimal cell In 2016, the same Craig Venter also presented a minimal cell. ‘Venter's group had taken a simple bacterium with a genome of around 1,000 genes and started taking bits out of it’, explains Dogterom. ‘After removing a piece of DNA, they would check to see if the cell was still functioning.’ By gradually shaving off the genetic material gene by gene, the group eventually discovered a minimum number of genes that the bacterium needed to keep itself alive. That magic number? 473 genes. Fewer than that and the cell no longer functioned. Craig Venter's ‘minimal bacterium’ is interesting, but also slightly surprising. Because if you look at the separate components that experts believe are needed in order to construct a complete cell from the bottom up, and the genes that code for them, 473 seems like a very large amount. Coordination and timing Christophe Danelon , who also conducts research into synthetic cells with his group in Delft, says he is aiming for a ‘global approach’. Danelon: ‘We do not focus on a single component, but we attempt to construct the different modules we need simultaneously.’ Not so long ago, for example, Danelon's group succeeded in artificially synthesising phospholipids. These fatty chemicals form the ‘vesicles’ in which the other parts of the synthetic cell will soon have to do their work. Danelon estimates that the components needed for a minimal cell can be made using approximately 150 genes, which is not even a third of Craig Venter's 473 genes. This therefore raises a question: will these 150 genes work together when they are combined into one singe strand of DNA? Danelon suspects that the genes in Venter's cell whose purpose is still unknown may be involved in such things as the coordination and timing of cellular processes. Ultimately, everything has to happen in the right sequence. For example, it makes no sense for a cell to start dividing if no copy has yet been made of the genetic material. Cutting through the middle Cees Dekker is the third leading TU Delft researcher involved in the project. Just like Marileen Dogterom, he has a strong physics background. The two scientists also share a refusal to be deterred by the complexity of the cell. ‘It is absolutely true that life is enormously complex’, says Dekker. ‘But does that mean we should say: it is so complicated, let's not even start? Or should we say instead: We see an opportunity to construct test systems and look at what we understand about them? The latter, in my view. I am a scientist because I want to understand how nature works.’ Dekker's group is focused on synthetic cell division, among other things. Some time ago, his group contributed to a study that demonstrated how the protein ring that cuts through the middle of a cell during cell division is constructed. Dekker: ‘I also conduct research into other biophysical processes, such as the spatial structure of DNA and proteins in cells. For example, we manipulated the shape of E. coli cells to see how DNA and proteins organise themselves.’ As well as providing some important scientific insights, this research resulted in the following extraordinary image: living bacteria in the form of the letters TUDELFT. Black box The Delft researchers emphasise that building a synthetic cell is above all a fundamental challenge. It is all about curiosity, about discovering how the cell works. But they are also convinced that the synthetic cell will result in all kinds of practical applications. ‘Just look at microbiology’, says Dekker. ‘In the beginning, research in that field was also driven by curiosity. But since then, a huge industry has emerged that uses bacteria as little factories to make all kinds of different substances.’ Marileen Dogterom expects that research into synthetic cells will result in medical breakthroughs: ‘When you administer a drug to someone, you are doing something with the molecules in their cells. Hopefully, that will have a favourable effect on the disease you are trying to combat. But in fact, you are putting those drugs into a kind of black box, because you do not know exactly what it is they do. This is why side effects are difficult to predict.’ The idea is that a better understanding of how cells work will enable us to intervene more effectively in the processes that make us ill. Ethical discussions Alongside practical applications, the construction of a synthetic cell is also likely to provide us with a greater understanding of what ‘life’ is, exactly, and how it began. ‘We have been doing cellular research for 70 years, and we have made tremendous progress’, says Dekker. ‘But no one knows how, four billion years ago, life first formed from separate biological components such as amino acids and lipids. That is a fascinating scientific question, and building a synthetic cell can help us find the answer.’ It may also raise ethical discussions on the issue of whether, as a scientist, you are permitted to ‘play God’: in other words, whether you should want to create life at all. ‘But’, says Dekker, himself a man of faith, ‘there is a perfectly good answer to that question. Moreover, it is doubtful whether the wider public will have such strong concerns about a primitive form of life. Currently, it is mainly journalists who raise the issue.’ Nevertheless, philosopher Hub Zwart from the Radboud University has now joined the Dutch consortium that has been awarded a prestigious NWO Gravitation grant for research into the synthetic cell. Linking pieces together How long will it be before the synthetic cell is a reality? It is difficult to say. ‘I am optimistic that, on a ten-year time scale, we will be able to make use of minimal components to make a system that can divide autonomously’, says Cees Dekker. Marileen Dogterom is slightly more tentative: ‘I am certain that it is possible to build a synthetic cell, but I am unsure whether that will be achieved within ten years. We are already capable of getting the pieces up and running. The great challenge is how we link these pieces together.’ Christophe Danelon loves his work and is quite happy for the research to take a long time. ‘I think I may have to sabotage the experiments when I sense that we are on the verge of a synthetic cell’, he says jokingly. But even if it proves possible to build a functioning, self-dividing cell, the question is whether that will conclude the project, says Dogterom. ‘Ultimately, it is about how you define success. For example, should a cell also be able to adapt, to evolve? What would you need to add to a synthetic cell to make that possible? And do we not also want to understand the interactions between cells?’ To ask the question is to answer it. And so, Christophe Danelon does not have to worry. There is more than enough work to do. Marileen Dogterom +31 (0)15 27 85937 M.Dogterom@tudelft.nl Cees Dekker +31 15 2786094 c.dekker@tudelft.nl group website Christophe Danelon +31 (0)15 27 88085 c.j.a.danelon@tudelft.nl This is a story from Applied Sciences Related stories Savings lives with mathematics Bacteriophages a possible alternative to antibiotics Tinkering under the bonnet of life
Tinkering under the bonnet of life
A longer life in good health? The end of genetic disorders? Crops that are able to survive in the harshest conditions? CRISPR-Cas9 brings all of this and more within our grasp. The research group of Dr Stan Brouns at the department of Bionanoscience is conducting fundamental research into how CRISPR systems function. What is his take on the forthcoming revolution? “Look, this is a Cascade complex,” says Dr Brouns, turning a strange-looking lump of plastic over in his hands. The object most resembles a chunk of coral, with a contorted and uneven surface. It is, in fact, a model of a cluster of proteins that are to be found in the cytoplasm of certain bacteria. “This protein here,” he points, “is the spine of the complex. And see these blobs here? Those are also proteins. All of them are essential, otherwise the system doesn’t work.” Molecular scissors What is the function of this particular cluster of proteins? Put simply, it is an essential weapon that bacteria use to exterminate viruses, which are professional killing machines that have been bacteria’s nemesis since the dawn of evolution. For example, every day, simple viruses known as ‘bacteriophages’ kill one third of all the bacteria in the oceans. “The virus injects its DNA or RNA into a cell in order to try to take it over,” explains Dr Brouns. “And if this hijacking is successful, the virus is able to use that cell as a little factory to produce copies of itself.” A strategy as simple as it is deadly. Dr Brouns’ protein cluster, the Cascade complex, leaps into action the moment a virus injects its genetic material. It seeks out the virus’s DNA and clamps onto it tightly. It then sends a signal to another protein, which comes along and cuts the virus to ribbons using a pair of molecular scissors. Dr Brouns is investigating how this type of bacterial defence system functions. Mysterious code How does the Cascade complex distinguish between viral DNA and the bacterium's own DNA? That is where something known as CRISPR comes in: ‘Clustered Regularly Interspaced Short Palindromic Repeats’. Researchers discovered these constantly repeating sequences of code (‘repeats’) in the DNA of the well-known E. coli bacteria in the 1980s. “Back then, nobody knew what the sequences meant,” says Dr Brouns. “It was only in 2005 that it was recognised that the DNA segments located between the repeats corresponded with the DNA of viruses.” The DNA of viruses? Yes, indeed. Sometimes a bacterium survives an attack by a virus, for example because the virus was weakened. In those rare cases, the bacterium seizes a trace of the virus’s DNA. By ‘unzipping’ its own DNA at the location of the repeats, the bacterium then builds the virus’s DNA into its own genetic code. The effect is comparable to how a vaccine works. From that moment on, the bacterium has that virus etched into its memory forever. If that same type of virus is foolish enough to try attacking that bacterium again, it recognises its assailant and can dispatch a cutting protein, such as the Cascade complex. Using two strands of RNA as a ‘cheat sheet’, the protein cluster goes on the hunt for enemy DNA. And if the complex finds a match, it is simply a matter of grip, snip and away. Cutting and pasting But CRISPR systems such as the Cascade complex are much more than just a biological curiosity. Scientists have been able to use them to edit DNA since the autumn of 2012, when Professors Jennifer Doudna (UC Berkeley, USA) and Emmanuelle Charpentier (then at Umeå University, Sweden) succeeded in reconstructing the CRISPR system of a streptococcus bacteria that uses the Cas9 protein. The two scientists unravelled the bacteria’s defence mechanism. Then, one of Prof. Doudna’s post-doc researchers managed to combine the two RNA stands into a single ‘guide RNA’ (the cheat sheet alluded to earlier), which he could programme however he wished. This made it possible to use any arbitrary segment of DNA as a guide RNA and therefore to cut DNA wherever required. Of course, rewriting DNA is quite a different challenge than merely cutting it apart. But further research also proved the latter to be possible using CRISPR-Cas9. Researchers are now able to send a segment of DNA along with the Cas9 protein. As soon as the protein snips through a cell’s DNA, the cell tries to repair the strand as quickly as possible. If the ends of the accompanying DNA segment fit with the ends of the cut strand, the cell will use that genetic material to repair the strand. Researchers are able to place other nucleotides (and even complete genes) between the ends of the accompanying DNA segment. Aggressive white blood cells CRISPR-Cas9 is now being used by thousands of research teams around the world to very precisely deactivate or overwrite particular genes. This technology has enormous potential. “One specific example is a clinical trial that is currently in progress in which white blood cells extracted from cancer patients are being modified using Cas9 to be more aggressive,” says Dr Brouns. “The hope is that this will enable the modified white blood cells to get rid of tumours on their own.” It is just one example of the numerous possible applications of CRISPR-Cas9. Naturally, the technology has certain limitations. “Gene editing in living creatures, for example, is a challenge,” he notes. Particularly when trying to introduce changes in tissue that is rarely regenerated. Consider, for example, Duchenne Muscular Dystrophy (DMD), which damages and weakens the muscles. “Setting aside the ethical objections for a moment,” says Dr Brouns, “in theory you could deactivate this disease at the embryonic stage. Or you could select embryos that are unaffected by this disorder, which already happens in clinics. But once a person is born with this disease, it is actually too late.” Still, scientists have already made significant strides towards a cure. “It is possible to embed a repair sequence into a specific virus and inject that virus into a mouse with DMD. The virus will then disseminate the new genetic material.” And the outcome of this procedure? Mice with stronger muscles. But unfortunately not as strong as those of healthy mice, as yet. A kit full of tools What is not widely known is that there are considerably more CRISPR systems in existence than merely the variant that uses the now familiar Cas9 protein. And the functioning of one can be quite unlike the next. For example, instead of a straight cut, one type of protein makes an angular incision in the DNA into which the repair material fits like a puzzle piece, which increases the cell’s chances of successfully repairing its DNA. Furthermore, the size of the Cas proteins varies in each system. Cas9 is relatively large and is therefore not able to operate in every type of cell. Another type of cutting enzyme, known as Cpf1, is also looking promising. “Cpf1 is going to be quite successful as well,” predicts Dr Brouns. “It has a tiny RNA that is capable of cutting through both DNA strands. And since it comprises only 40 nucleotides, compared to Cas9's 100, it is cheaper to produce. Moreover, it makes it easier to cut in multiple locations simultaneously.” In short, CRISPR-Cas is not a single tool, but rather a whole kit full of different kinds of implements. Where one job requires a screwdriver, another requires a hammer. And the good news is, new tools are being added to the kit all the time. Hopefully these new discoveries will eventually lead to a point where scientists can mould all extant cells to their will. Breaking new ground And where will all this end? The first children with a modified DNA have already been born. And while we are at it, will we choose to make these genetically modified children more intelligent? With violet eyes? And maybe add some sweet little dimples to their cheeks? “Yes, well,” says Dr Brouns, “at a certain point you stray beyond the bounds of diseases into areas that people find desirable. That is something we have to consider with great care.” Brouns knows, of course, that it is becoming more and more pressing to answer questions of this nature. However, his goal is to unravel the biology behind CRISPR-Cas systems in order to figure out down to the minutest detail how these extraordinary defence mechanisms function. One question that the Brouns group is now trying to answer, for instance, is why bacteria seem to prefer taking certain strands of a virus’s DNA to build into their own DNA, as opposed to others. These endeavours will keep Brouns and his research group occupied for some time yet. “We have already learned so much, yet I expect that there are many microbial defence mechanisms still to discover.” But the most striking thing remains just how ingenious the CRISPR systems are, which is precisely why they are a source of fascination. “Every time we look at them, their functioning always seems more complex than we first thought,” says Dr Brouns. “They never cease to amaze us.” Stan Brouns +31 15 27 83920 S.J.J.Brouns@tudelft.nl This is a story from Applied Sciences Read more about this project “Look, this is a Cascade complex,” says Dr Brouns, turning a strange-looking lump of plastic over in his hands. The object most resembles a chunk of coral, with a contorted and uneven surface. It is, in fact, a model of a cluster of proteins that are to be found in the cytoplasm of certain bacteria. “This protein here,” he points, “is the spine of the complex. And see these blobs here? Those are also proteins. All of them are essential, otherwise the system doesn’t work.” Molecular scissors What is the function of this particular cluster of proteins? Put simply, it is an essential weapon that bacteria use to exterminate viruses, which are professional killing machines that have been bacteria’s nemesis since the dawn of evolution. For example, every day, simple viruses known as ‘bacteriophages’ kill one third of all the bacteria in the oceans. “The virus injects its DNA or RNA into a cell in order to try to take it over,” explains Dr Brouns. “And if this hijacking is successful, the virus is able to use that cell as a little factory to produce copies of itself.” A strategy as simple as it is deadly. Dr Brouns’ protein cluster, the Cascade complex, leaps into action the moment a virus injects its genetic material. It seeks out the virus’s DNA and clamps onto it tightly. It then sends a signal to another protein, which comes along and cuts the virus to ribbons using a pair of molecular scissors. Dr Brouns is investigating how this type of bacterial defence system functions. Mysterious code How does the Cascade complex distinguish between viral DNA and the bacterium's own DNA? That is where something known as CRISPR comes in: ‘Clustered Regularly Interspaced Short Palindromic Repeats’. Researchers discovered these constantly repeating sequences of code (‘repeats’) in the DNA of the well-known E. coli bacteria in the 1980s. “Back then, nobody knew what the sequences meant,” says Dr Brouns. “It was only in 2005 that it was recognised that the DNA segments located between the repeats corresponded with the DNA of viruses.” The DNA of viruses? Yes, indeed. Sometimes a bacterium survives an attack by a virus, for example because the virus was weakened. In those rare cases, the bacterium seizes a trace of the virus’s DNA. By ‘unzipping’ its own DNA at the location of the repeats, the bacterium then builds the virus’s DNA into its own genetic code. The effect is comparable to how a vaccine works. From that moment on, the bacterium has that virus etched into its memory forever. If that same type of virus is foolish enough to try attacking that bacterium again, it recognises its assailant and can dispatch a cutting protein, such as the Cascade complex. Using two strands of RNA as a ‘cheat sheet’, the protein cluster goes on the hunt for enemy DNA. And if the complex finds a match, it is simply a matter of grip, snip and away. Cutting and pasting But CRISPR systems such as the Cascade complex are much more than just a biological curiosity. Scientists have been able to use them to edit DNA since the autumn of 2012, when Professors Jennifer Doudna (UC Berkeley, USA) and Emmanuelle Charpentier (then at Umeå University, Sweden) succeeded in reconstructing the CRISPR system of a streptococcus bacteria that uses the Cas9 protein. The two scientists unravelled the bacteria’s defence mechanism. Then, one of Prof. Doudna’s post-doc researchers managed to combine the two RNA stands into a single ‘guide RNA’ (the cheat sheet alluded to earlier), which he could programme however he wished. This made it possible to use any arbitrary segment of DNA as a guide RNA and therefore to cut DNA wherever required. Of course, rewriting DNA is quite a different challenge than merely cutting it apart. But further research also proved the latter to be possible using CRISPR-Cas9. Researchers are now able to send a segment of DNA along with the Cas9 protein. As soon as the protein snips through a cell’s DNA, the cell tries to repair the strand as quickly as possible. If the ends of the accompanying DNA segment fit with the ends of the cut strand, the cell will use that genetic material to repair the strand. Researchers are able to place other nucleotides (and even complete genes) between the ends of the accompanying DNA segment. Aggressive white blood cells CRISPR-Cas9 is now being used by thousands of research teams around the world to very precisely deactivate or overwrite particular genes. This technology has enormous potential. “One specific example is a clinical trial that is currently in progress in which white blood cells extracted from cancer patients are being modified using Cas9 to be more aggressive,” says Dr Brouns. “The hope is that this will enable the modified white blood cells to get rid of tumours on their own.” It is just one example of the numerous possible applications of CRISPR-Cas9. Naturally, the technology has certain limitations. “Gene editing in living creatures, for example, is a challenge,” he notes. Particularly when trying to introduce changes in tissue that is rarely regenerated. Consider, for example, Duchenne Muscular Dystrophy (DMD), which damages and weakens the muscles. “Setting aside the ethical objections for a moment,” says Dr Brouns, “in theory you could deactivate this disease at the embryonic stage. Or you could select embryos that are unaffected by this disorder, which already happens in clinics. But once a person is born with this disease, it is actually too late.” Still, scientists have already made significant strides towards a cure. “It is possible to embed a repair sequence into a specific virus and inject that virus into a mouse with DMD. The virus will then disseminate the new genetic material.” And the outcome of this procedure? Mice with stronger muscles. But unfortunately not as strong as those of healthy mice, as yet. A kit full of tools What is not widely known is that there are considerably more CRISPR systems in existence than merely the variant that uses the now familiar Cas9 protein. And the functioning of one can be quite unlike the next. For example, instead of a straight cut, one type of protein makes an angular incision in the DNA into which the repair material fits like a puzzle piece, which increases the cell’s chances of successfully repairing its DNA. Furthermore, the size of the Cas proteins varies in each system. Cas9 is relatively large and is therefore not able to operate in every type of cell. Another type of cutting enzyme, known as Cpf1, is also looking promising. “Cpf1 is going to be quite successful as well,” predicts Dr Brouns. “It has a tiny RNA that is capable of cutting through both DNA strands. And since it comprises only 40 nucleotides, compared to Cas9's 100, it is cheaper to produce. Moreover, it makes it easier to cut in multiple locations simultaneously.” In short, CRISPR-Cas is not a single tool, but rather a whole kit full of different kinds of implements. Where one job requires a screwdriver, another requires a hammer. And the good news is, new tools are being added to the kit all the time. Hopefully these new discoveries will eventually lead to a point where scientists can mould all extant cells to their will. Breaking new ground And where will all this end? The first children with a modified DNA have already been born. And while we are at it, will we choose to make these genetically modified children more intelligent? With violet eyes? And maybe add some sweet little dimples to their cheeks? “Yes, well,” says Dr Brouns, “at a certain point you stray beyond the bounds of diseases into areas that people find desirable. That is something we have to consider with great care.” Brouns knows, of course, that it is becoming more and more pressing to answer questions of this nature. However, his goal is to unravel the biology behind CRISPR-Cas systems in order to figure out down to the minutest detail how these extraordinary defence mechanisms function. One question that the Brouns group is now trying to answer, for instance, is why bacteria seem to prefer taking certain strands of a virus’s DNA to build into their own DNA, as opposed to others. These endeavours will keep Brouns and his research group occupied for some time yet. “We have already learned so much, yet I expect that there are many microbial defence mechanisms still to discover.” But the most striking thing remains just how ingenious the CRISPR systems are, which is precisely why they are a source of fascination. “Every time we look at them, their functioning always seems more complex than we first thought,” says Dr Brouns. “They never cease to amaze us.” Stan Brouns +31 15 27 83920 S.J.J.Brouns@tudelft.nl This is a story from Applied Sciences Related stories Saving lifes with mathematics Bacteriophages as a possible alternative to antibiotics Constructing living cells
