What are genomic scissors
Why is CRISPR/Cas9 called the grail of modern medicine?
One of the most promising methods of treating cancer and other terrible diseases is genome editing. This technology is called CRISPR/Cas9. What is its essence, says Konstantin Severinov, a specialist in the regulation of bacterial gene transcription, professor at the Skolkovo Institute of Science and Technology, Professor at Rutgers University (New Jersey, USA), head of laboratories at the Institute of Molecular Genetics of the Russian Academy of Sciences and the Institute of Gene Biology of the Russian Academy of Sciences.
I would like to tell you about the bacterial immunity system, which is called CRISPR-Cas, and in addition to such a small fundamental introduction about what it is, why it is needed in nature, to tell you why we, people, could need it, what medical applications, biotechnological applications, environmental, whatever.
As an introduction, we should imagine that our planet is a planet of bacteria, and not people at all. Here I have so conditionally drawn a bacterial cell that lives, divides and, in general, bacteria have invaded the whole earth, and not people at all. Moreover, bacteria have invaded, in particular, people, so there are more bacterial cells inside you and me than there are actually our cells with you.
But it would be wrong to assume that bacteria thereby rule the world, because in fact bacteria have a huge number of viruses and the number of these viruses that parasitize bacteria is absolutely monstrous, that is, someone thought that there are 10 to 31 degrees of viral particles on Earth. This is some absolutely phenomenal amount, well, just a lot. And viruses have been killing bacteria for billions of years, during which life has existed, and they do it very effectively.
Here I draw such a viral particle very schematically, it looks like a syringe. Inside this viral particle, in this head there is viral DNA, deoxyribonucleic acid, genes. Viruses have genes and this is what happens.
After the viral particle recognizes the cell, it recognizes it according to the key-lock principle (yes, each virus can recognize its corresponding cell), then the viral DNA is "injected" into the cell, and then this DNA begins to multiply, and becomes more and more, the cell is infected. It's a bit like what happens to us when we get the flu.
At the same time, the genes that are on the viral DNA are synthesized into special proteins, viral, in particular, necessary in order to create still empty viral particles, and then the viral DNA is packed into a viral particle and suddenly, literally 10 minutes later, the bacterial cell burst, all its contents flowed out, but outside daughter viral particles also came out, which are completely identical to the virus that infected the cell, just their number has increased dramatically.
As a result, 15-20 minutes after the initial recognition of the host bacterial cell by a viral particle, the bacterial cell bursts, actually about a hundred new viral particles come out, which are able to repeat the whole cycle, yes. And so it happens all the time.
And we must imagine that if such a process really happened all the time, then very soon there would be no bacteria in the world, because bacteria do not have time to divide at such a speed to compensate for their death as a result of the development of viruses, viruses are becoming more and more.
Obviously, this is not happening and the question arises: why? Why do bacteria exist? That's an interesting question. Not necessarily bad bacteria, there are also good bacteria.
For every bacterial cell in nature, well, again, these estimates are all very approximate, there are about 10 viruses. Therefore, in principle, all bacteria must die, and then viruses must also die, because they become alive only insofar as there is a cell in which they exist. In a free external environment, they are, in general, not very durable.
It would seem that everything should have stopped, and the game would have ended, but of course it continues and will continue for a long time after you and I disappear from the face of the planet.
The answer to the question of why bacteria exist is that they have protective systems. They're not fools either. They have defensive systems. Of course, the Lord God did not give it to them, they just acquired it in the course of evolution. A special device, gadgets, devices, whatever you want, that allow them to fight viruses. Those bacteria that don't have such devices, they really died and therefore we don't see them anymore. Such a typical Darwinian evolution.
One of these protective systems is this very CRISPR-Cas, it's just one of the protective systems with which bacteria fight viruses. They opened this system literally 15 years ago there. How does it work? Let's draw a bacterial cell again, we'll have a bigger one now, because we'll look inside it, we need to figure out what's going on here. After all, bacteria also have DNA, there is DNA in our cells, bacteria have DNA, and viruses, as we have just found out, have DNA. Let it be the DNA of a bacterium, which contains numerous genes that bacteria need in order to exist.
If a virus has joined our cell and injected its own viral DNA into it, these are DNA viruses. By the way, such viruses are usually called bacteriophages in the case of bacteria, because they literally devour bacteria, a phage is a virus eater, a bacteria eater.
It turns out that a cell can sometimes, if a cell has this CRISPR-Cas protection system, it has, somewhere inside this bacterial chromosome of its DNA, a special section called CRISPR and this is just an abbreviation from an English long scientific term. But what is important for us is that this site is actually a memory.
What is our memory? Memory is a certain place in our brain, we really don't know where it is, where we put some memories, like in such a cache, everything that happened to us, everything that we read and then we can use it if we need it.
So it turns out that small pieces of viral DNA can be selected and inserted here into this bacterial memory. If such a process had occurred, and in the CRISPR-Cas system it just happens, there would have been an insertion of a small piece of virus-infecting bacterial DNA.
In most cases, this is a random process. Of course, it is regulated by proteins, everything that happens in the cell is controlled by genes, in the sense that genes encode proteins that do something. These are proteins, enzymes that, as it were, allow us to be what we are, absorb food, produce some substances, and so on. Specifically, bacteria that have the CRISPR-Cas system have a set of genes whose products sometimes allow them to recognize a random fragment of viral DNA and insert it into their memory.
The amazing thing is that if such a piece is inserted inside the cellular DNA, then the bacterium acquires the ability to fight the virus, but not with any virus, but only with the virus that has a site corresponding to this memory.
If a cell was able to remember the virus by the fact that a small piece of viral DNA was inserted into its memory area, then further in such cells, if they have a CRISPR-Cas system, there is a protein, we will call it Cas9, no matter why it is called that, but it is, in essence, molecular scissors. There is a special gene here. This is the Cas 9 gene, a section of DNA that encodes such a protein.
Protein is just molecular scissors, but they are not bad scissors that bite everything, they bite only the very right things. In fact, this protein receives information about the piece of viral DNA that has been inserted into memory. We will call this piece a guide. Just like when you go to some unknown city, you can hire a guide who will show you everything.
And when our scissors contacted such a guide, now these scissors have acquired the ability to programmatically recognize the viral DNA that corresponds to the guide and no other. And after this recognition occurs, we cut this viral DNA with our scissors. Not here, not here, but exactly in the place that corresponds to the guide.
Well, of course, after the viral DNA was cut, then such an infection process will not go. At the earliest stage of infection, only when the viral DNA enters the cell, it will be cut, and the cell will survive. And the cell remembered it, as it inserted this memory, like a vaccination book, a medical book, its own DNA, then all the descendants of this cell will also remember it.
First, the guide got from the virus into memory, then this memory is realized, because your memory also does not necessarily always function by itself, so you remembered me only because you saw me, yes, here. And it's the same here, that is, if, in order for this memory to work, it is necessary that the guide gets to the Cas 9 protein and now these scissors have acquired some meaning.
Previously, they were such completely meaningless scissors, and now they are armed, they have such, well, you want, a passport. Consider that this is a policeman and he has an objective that someone needs to be caught. Whom? The one who is like this. If such a person suddenly gets here, we will immediately kill him. Hello, everyone. Well, the cells live, here.
Obviously, viruses have their own ways to deal with this, otherwise viruses would have died out. After all, when I draw this picture for you, it is clear that if bacteria have to escape from viruses and kill them, then viruses must somehow participate in this war, win the arms race.
Viruses have anti-CRISPR systems. Many viruses, for example, take, and the Cas9 protein is killed. They have a special way when they, along with their DNA, inject another special protein into the cell, just in case, which Cas 9 kills. Well, of course, then the cage is bad, you need to come up with something else.
But in principle, we don't really care about everything. Or rather, I didn't care until a certain period, that is, why CRISPR-Cas is what everyone is talking about now, it's not at all related to the fact that we are interested in how bacteria fight viruses there, we are interested, of course, in ourselves, we are relatives.
So what is genomic editing anyway? This is, in general, genomic editing, the Holy Grail of medicine. The fact is that there are a lot of diseases that are associated with the fact that some kind of change, hereditary or acquired, has occurred in the DNA sequence of the patient's cells, and it is the sequence of letters that has changed, these A, G, C, T, they have somehow changed.
So let's draw a DNA chain, well, not a DNA chain, but this famous double-stranded spiral. And imagine, here is one chain, here is another, this is a very short section really, of course it will be huge, in terms of the number of turns and information. And here there is a certain sequence of letters: A, G, C ... These are chemical letters, these are not real letters, but chemists, biologists simply prefer to call these chemical groups letters.
And there is a certain rule according to which there is a mapping rule opposite this blue chain, when there will always be T against A, against G, C, and so on and so forth. Everyone knows this or should know it from school, and if they don't, then they should be ashamed.
And let the majority of healthy people have a DNA sequence exactly in this place, but no other. And let this section of DNA encode some important protein, for example, a protein whose activity does not allow tumors to develop, well, in a normal state.
A mutation can occur, a mutation is just a DNA change. Look, in a normal situation here we have the letters C, G, and so it happened, someone was very unlucky that the letters A, T became here. This mutation is in the sense that the state of DNA in this particular position is not normal.
Changing the genetic information in the form of a DNA sequence leads to the fact that you have some kind of damaged protein, which leads to one or another undesirable consequence. I must say that, generally speaking, this is a rare event, because for the most part such changes do not lead to anything.
What should we do if we want to treat this unfortunate? You can come up with some medications that act on this bad protein, you can come up with medications that act on bad cells, or you can call a surgeon to cut something out for you there. But in principle, the best way is genomic editing, the most like this, well, there's nowhere else, namely.
If we could, as genomic, as genetic surgeons or DNA surgeons, find this place in the cells of the tissue that is damaged as a result of such a mutation, as on an old typewriter to erase these wrong letters, and put the right option here, then the cause of the disease disappeared. We would have returned to a really normal state. The principle is very simple.
There is only one problem: to do this, you need to find and change the letter in exactly the right place. We are talking about changing one letter on a relatively small piece of DNA.
But the fact is that the size of our DNA, the size of the DNA of a human cell, is 3 by 10 in the ninth, that is, 3 billion letters, such a long, long chain. To understand how much it is, a wonderful book "War and Peace" by Leo Tolstoy, which, as all schoolchildren know, is thick, it contains literally a couple of million letters. That is, there are thousands of "Wars and Worlds" here. And it says that you are you, and I am me.
Moreover, we get this only from mom, we get the same amount from dad, we have two sets of chromosomes and two sets of genes, we have all the genes copied. That is, we have to find this one change in a stack of books, in a thousand books the size of "War and Peace", find this typo, read it somehow very quickly and change it.
And at the same time, it is advisable to do this exactly so as not to make any "mistakes" in other places, because then you will only make it worse. How would we do that? In principle, how to do this is clear, because, look, I wrote that we have 3 by ten in the ninth letter of DNA.
We have 23 chromosomes from dad, 23 chromosomes from mom, each chromosome is just a long DNA molecule. How long is it? How much, how long will such a book of billions of sequences of letters A-G-C-T be, given that of course DNA is just a tiny molecule. It turns out that it will be a very long molecule, if you take the DNA from each of my cells, put it on the butt and straighten it, then it will be about as tall as me, a long thing like that. From every cell. And I have trillions of cells in total. In this sense, it is necessary to treat your body very respectfully, it is generally a completely wonderful machine. And the cells, of course, divide. Every time a cell divides, the DNA in it doubles and then one, one set of all the genes, and all the DNA molecules go into one cell, another into another.
Imagine the task when you have such a thin thread as long as my height, tangled, somehow packed like this into a tiny cell that is not even visible to the eye, and now you have to copy, make a copy of this thread and then divide it into two cells. With a high probability, your DNA will be torn. If you try to pull it apart, it will tear at you, and it really happens. DNA is always torn, especially during cell division.
This, in fact, is one of the reasons why in the course of life we eventually acquire cancer. Because we struggle with these gaps, cells have learned to fight them, but sometimes they make mistakes.
So, an important statement. Cells are able to fight DNA breaks, all cells. Those cells that don't know how die. Cells are able to fight DNA breaks. And DNA breaks always happen, you just have to come to terms with it. With DNA breaks.
How is this done? And here's how. Imagine that this is Dad's DNA molecule, and this is the corresponding mom's DNA molecule, and let the gap happen here by chance. Generally speaking, a disaster, just the DNA broke. But we do know that these two molecules are the same, yes. It's from Dad, it's from Mom, but they're basically the same.
It turns out that there are protection systems in the cage against such randomly occurring breaks. How? Namely, because we have this is a copy of this, we can restore the gap from the unaffected copy. This is happening, those who still remember, it probably looks like, probably to some extent similar to this tape that used to be on the cassette, it was torn, so, and you can just connect it joint to joint, or you can just overlay a piece from another version and restore everything.
I will draw it in such a way that here I seem to have had an overlap, and I have healed this gap on this healthy copy. Those cells that don't know how to do that, they get sick and die, and they don't really exist. Those people who do not have special proteins that can do this, they are usually photosensitive, in general, they are also very unhealthy, because light, just sunlight causes us to break DNA, by itself. That is, we must be able to sew up these gaps.
But then there is an idea of how to link this with genomic editing. Namely, imagine that you have a genetic disease, even if it's not you, even if it's me, even if it's someone else, someone we don't know. And this genetic disease arises from the fact that the copy of the gene that came from Dad contains a change here, in the spirit of what we had here, yes. And Mom's copy is normal, yes. A person has one defective copy of the gene and this makes him very unhappy and sick.
The situation when a person has two defective copies of the gene from both mom and dad is very rare, and, as a rule, such people are not even born, they die in utero. Since we know that a cell can heal DNA, it is very easy to assume how we can deal with it in principle.
We need to figure it out somehow, so if we had scissors, if we had such a miracle and we could introduce a gap into the very place where the mutation that causes us problems occurred, then what would happen? The cell will consider it simply as a break in the DNA, it knows how to treat it, and it will heal this gap on a healthy copy in this way, yes. Everything is fine, we are cured.
Then our problem of genomic medicine now becomes somewhat different, we can ask the question in a different way: how is it directed or programmed to introduce breaks in DNA? After all, if we could do this, we could treat genetic diseases. Because the cell already knows how to sew up breaks that happen by chance. All we need, as scientists, doctors, is to learn how to introduce these gaps not just where, but where we are interested.
Where are we interested? Where there are mutations that lead to some kind of genetic diseases. How do we know where these mutations are? Because we have the Human Genome project. More and more genomes are determined by it, doctors little by little find all those changes that are responsible for this or that disease.
Now we have the third part of the lecture and it's about how we tie the first two together. You and I know that bacteria can fight viruses using the CRISPR-Cas system when they remember the virus, and then the recognized viral DNA is cut using programmable scissors. What they remember, they will cut, yes. And what they don't remember, they can't cut. And on the other hand, you and I know that there are genetic diseases that, in principle, could be cured if we could make a break in the DNA at the place where the mutation occurred. Then it's a matter of technique, the cell itself knows how to repair the gap on a healthy copy, everything is fine.
How are these two things related? On the one hand – bacteria, on the other hand – serious problems associated with genetic diseases. Cancer, psychological diseases, cystic fibrosis, blood disease, whatever. And here's how.
In our initial situation, our molecular scissors Cas 9, this is a protein from bacteria, note, yes, it is programmed by a guide that corresponds to a section of viral DNA, viral DNA in order to cut viral DNA, and this is the biological meaning.
And let's spit on biology, biotechnologists always spit on biology. Let's program our protein with a guide whose sequence did not come from a virus, but whose sequence corresponds to an altered copy of a human gene of some kind.
It turns out to be very simple. Because a guide is just a piece of nucleic acid, it's very easy to make. Then we should see this. Let's draw again two corresponding DNA molecules of a human cell from dad, from mom, and let us have some genetic, some kind of aberration, mutation here that we need to cure.
Then, if my guide is programmed to recognize this and only this copy (and the guide knows how to point scissors at certain sequences), this difference here is one, one letter is enough for him not to recognize this copy, but to recognize this one.
What will happen? Our scissors, having learned this version of the sequence, will figure it out, we don't need Cas9 for anything else, because everything else will happen by itself, because the cell has learned to do this during thousands, billions of years of evolution.
Either the cell will die, nothing will happen in a number of cases, or a repair will simply take place and as a result, this gap will be patched up for this healthy copy. And, consequently, as a result of such genomic editing, we have programmatically changed the DNA sequence. If we had one copy of DNA, one chromosome is normal and the other is mutant, then now they are both normal.
It happened, we overcame some very different barriers, we started with biology and virus protection. We must understand that we are in a human cell now, all this happens inside a human cell, and maybe even inside a human egg, as in fact in these famous experiments, recent, by a Chinese scientist who edited, well, future children.
He took an egg, it has, of course, chromosomes, genes, and so on. And with the help of this technology, I introduced a raskus in a certain place and changed one of the versions of chromosomes. Well, then a man appeared, because after such a change occurred, the cell divides, everything goes fine, a man appears.
We have introduced a bacterial protein into a human cell, this is the first thing to understand. We programmed this protein not with what it is usually programmed with, not with a piece that protects against viral DNA, from bacteria, but with a site that is specific, which recognizes a certain fragment of human DNA that is interesting to us.
And then, in an amazing way, the bacterial protein worked in the human cell in the same way as it usually works in a bacterial cell. Bad programmable scissors, what you program them for, the perfect performer, will bite there. Then everything went like clockwork.
And it is clear that the procedure is so general that you can edit both human cells and people themselves, if you do it on embryos, you can edit plants, you can edit farm animals, if you are interested in changing their genetic sequences in some way, you can edit bacteria, you can do anything. Another question is, you need to know what you're doing. And this is both the strength and the limitation of this method.
In most cases, we don't know which change in DNA is responsible for which property. That is, if for a number of serious diseases, for example, for cystic fibrosis, it is known exactly where there is a change, and it is clear exactly what can be treated, then for properties like intelligence, height, beauty, whatever it means, yes, the shape of the nose and so on, there are no such changes, such changes in DNA it is known. We don't know what it is.
And in this sense, to be afraid of the fact that now everyone will start making such designer children, as in the menu, when you come and order: give me a twist here, here and here, we are very far from this, and most likely we will never come to this, simply because we don't know it.
But for the treatment of relatively simple diseases caused by changes in understandable genes that have been identified by medical geneticists, well, over the past hundred years, here, probably, there may be a breakthrough in the near future.
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