Genome in editing mode

Interview with Dmitry Zharkov

Anastasia Penzina, "Scientific Russia"

The Nobel Prize in Chemistry in 2020 was awarded to Jennifer Doudna and Emmanuel Charpentier for the discovery of the CRISPR-Cas gene editing system. This was a real breakthrough in applied biology and medical genetics. At the same time, technologies for directed modification of the genomes of living organisms have existed before. Genetic engineering as a scientific discipline has at least half a century of history. However, the previously existing methods required a lot of effort and time, and success was not guaranteed. Our conversation with Dmitry Zharkov is about the significance of the discovery awarded the Nobel Prize in Chemistry, about the ethical challenges facing modern genetics and synthetic biology.

Dmitry Olegovich Zharkov – Doctor of Biological Sciences, Director of the Center "Design of Living Systems" of Novosibirsk State University, corresponding member of the Russian Academy of Sciences.

– In 2020, the Nobel Prize in Chemistry was awarded to scientists Emmanuel Charpentier from France and Jennifer Doudna from the United States of America for the development of the genome editing method. At the same time, the CRISPR-Cas system, as far as I know, was known earlier, but it was Charpentier and Dudna who discovered another important property of this system – the ability to crack DNA like scissors in any sequence. How did they manage it and how did they come to it at all?

– Everything is actually a little more complicated. Indeed, the CRISPR-Cas system itself was not discovered by Dudna and Charpentier. For the first time, these repeat cassettes, which are called CRISPR (from the English clustered regularly interspaced short palindromic repeats – groups of short palindromic repeats separated by regular intervals. – Approx. HP), were discovered in 1987 by a group of Japanese scientists led by Atsuo Nakata in the bacterium Escherichia coli, in other words, E. coli.

Then in 2005, scientists showed that these repeating elements originate from the genomes of bacteriophages and plasmids, that is, molecular parasites of bacteria. Further in 2007, it was shown that the presence of these fragments, as well as adjacent genes, which were called Cas (Cas = C RISPR-A ssociated G enes, "CRISPR-associated genes". Associated – because they are close to CRISPR repeats. – Note by D.O. Zharkova.) – it is necessary to protect the bacterium from infection and the effects of foreign genetic elements – bacteriophages and plasmids.

Only after that, in fact, the study of the mechanism itself began. It is clear that protection against foreign DNA can be built in different ways. It can, for example, be bound or split. The merit of Dudna and Charpentier in this case is that they showed that the Cas9 gene – it belongs to the so-called type II CRISPR systems, which are not found in all bacteria – in fact, is the only protein of this system that is needed for the cleavage of foreign DNA. In the presence of two RNAs: sgRNA or guide RNA and tracrRNA, that is, trans-activating RNA, the protein becomes active and begins to cleave foreign DNA.

– How significant was the contribution of Dudna and Charpentier to the development of genetics?

– The contribution, of course, is huge. CRISPR-Cas technology has become the most breakthrough technology that appeared in the arsenal of genetic research after the discovery of the polymerase chain reaction in the 80s. Literally thousands of laboratories around the world use it. There are several dozen similar laboratories in Russia.

CRISPR-Cas technology has dramatically facilitated the processes of genome modification at the level of whole cells and living organisms. By that time, methods of working with isolated genetic elements – plasmids - were well developed in genetics. However, the genome design could be carried out, but with great difficulty. Traditional technologies for producing transgenic animals were based on a process called homologous recombination. Edited to the desired sequence could be one cell in a million. In the case of CRISPR-Cas technology, the number of edited cells reaches several tens of percent.

– In 2018, another significant event shocked the world – a professor from China announced that he had managed to edit the human genome. He Jiankui claimed that the girls born were immune to HIV. What do you think about these studies?

– For the scientific community, the value of this experiment is approximately zero, since the results have not been published. Gentlemen have not been taken at our word for a long time.

Everything we know about the experiment is known only from the words of He Jiankui himself, who in China received three years in prison for operations not provided for by law.

In principle, the technology already today allows you to change the genome in mice and even in humans. So the professor from China didn't really do anything new. However, a large layer of ethical issues has arisen. Do we have the moral right to interfere with the human genome? Will the changes lead to some unplanned consequences? Today, the only way to make sure that the necessary change has been made to the genome and no other changes have been made is to carry out a complete sequencing of this genome. He Jiankui stated that he had done a complete sequencing on cells from amniotic (amniotic) fluid. This fluid is tested to detect genetic diseases in the fetus. The task is solvable, but extremely expensive.

Today, the minimum cost of complete genome sequencing is measured in thousands of dollars. Therefore, a large-scale application of the editing method is impossible, since we simply do not know if any other changes have been made. And if we find out about it in the later stages of a person's life, it will be too late to treat him.

Therefore, we cannot evaluate the statement of the Chinese professor, even if he has stirred up this anthill, because there are no materials. The big question is, is it worth editing these genes? The CCR5 gene, which was the target in this case, has long been considered as one of the main candidates for editing the human genome. It encodes one of the main surface proteins of cells-lymphocytes, and the virus binds to it to enter the cell. At the same time, CCR5 has two variants (alleles) – conditionally normal and mutant, which almost does not affect human health, and even, perhaps, positively affects brain development, but this has not yet been fully proven. The virus does not bind to the mutant protein of the variant and penetrates the cells much worse. The question arises, is it worth influencing the human genome and treating it for AIDS in this way, when there are more traditional ways to protect yourself from infection?

On the other hand, it is clear that there are genetic diseases that we cannot prevent otherwise than by correcting the human genome. But here, too, it all comes down to ethics.

– Is it possible to predict the consequences of interference in the human genome?

– Good question. This is the path of the development of science – we are gradually moving towards the possibility of predicting the consequences of something. In the case of the genome, most likely, we will never fully learn how to determine them, simply because of the very complex network of relationships that exists in any human cell, between cells in the body, and so on. It is important to know where to stay here.

In fact, we never know one hundred percent what the consequences of using any medicine will be. But this does not prevent us from using them. In this case, we also need to define some kind of boundary where the use of extraordinary methods will be justified and safe. But the risk always remains.

– There is a widespread opinion, or even a metaphor, that DNA is a kind of text that geneticists literally read. How appropriate is this comparison?

– This is not even a metaphor, but one of the perfectly legitimate approaches to DNA analysis. When I tell students about this, I usually scold this approach, because in fact, of course, there is no text in DNA, but there are atoms and chemical groups.  But for the purposes of, say, bioinformatics, when we analyze the preservation of genetic information, evolution, and so on, this approach is absolutely legitimate. When we look at the physics of the interaction of proteins with DNA or DNA strands with each other, this approach is no longer sufficient, and we need to look for something else. But, in principle, yes, this is one of the possible, but not complete descriptions.

– When they talk about genome editing, personalized medicine is often mentioned. How are these concepts related?

– In fact, personalized medicine is already gradually becoming a thing of the past. Now another term is in vogue – "precision medicine". For example, personalized medicine takes into account the genetic composition of a person, that is, his genotype. Precision medicine, in addition, takes into account the condition of a person at any given moment. Relatively speaking, a person during the day and a person at night are always slightly different people, since the hormonal background changes, the systems of some organs work differently. And, in a good way, all this can be taken into account, but it is not clear whether it is necessary.

On the basis of genetic editing, different types of therapy are being tested, for example, oncological diseases. They are based on the fact that immune cells are taken from the patient, edited and injected back into them so that they more successfully recognize the antigens of the tumor of this particular patient.

But again, the main issue of personalized and precision medicine is not in technical capabilities, but in economics. That is, you need to understand that all these approaches are not even an order of magnitude, but many orders of magnitude more expensive than traditional ones. Treatment, personally tailored to a particular person, can be extremely effective, but it costs tens and hundreds of millions of dollars, and it is clear that without some general change in the approach to such types of therapy, we will not introduce them into wide circulation in any way.

– That is, while doctors continue to treat people with traditional methods?

– Of course, doctors will save people's lives by traditional methods for a long time. Thank them very much for this. But medical and genetic technologies are not standing still. The good news is that all types of genomic therapy and genomic editing have the property of universality. Take at least the CRISPR-Cas technology. In fact, it does not need to be changed when it is necessary to include gene A or gene B. Unlike traditional pharmacology, where each target protein requires its own active molecule, and the development of each of the drugs takes many years and costs a lot of money. In the case of CRISPR-Cas, it is enough to change the small guide RNA and continue working with another gene. And this is an advantage that, of course, the development of gene therapy will be associated with in the future.

– Let's talk about your research. Recently, together with colleagues from the Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences and Novosibirsk State University, you created a cocktail of proteins to work with degraded DNA. Tell us more about this study and what did you manage to achieve during its implementation?

– The study is not finished yet. In this case, the degraded DNA allows you to accumulate data for subsequent analysis. This is an urgent task, especially for the study of ancient DNA fossils. In addition, this method is suitable for forensic medical examination, for the analysis of the origin of DNA in processed foods, DNA analysis from medical archival samples that were once preserved.

For many years we have been working with DNA repair – a complex of many enzymes that protect us from mutation. Because human DNA is constantly being damaged, hundreds of thousands of damages occur in one cell every day. At the same time, they do not turn into mutations, the cell does not die, just because we have an active repair system. But after death, it is clear that the systems stop working and DNA begins to gradually decompose, oxidize, hydrolyze. Experts say that the natural limit of the existence of DNA outside the body does not exceed about a hundred thousand years. Everything that is ancient, we practically cannot pull out and analyze from these fossils in any way.

Therefore, the next reports that it was possible to find bacteria that had lain somewhere in amber or in a salt crystal for forty million years, as a rule, turned out to be unreliable when checked. So, knowing how the repair system works in the human body, in bacteria, and so on, we made our own mixture of several repair enzymes. Together with colleagues from the Institute of Molecular and Cellular Biology SB RAS, we tested it on some ancient samples, the age of which reached forty thousand years. The older the DNA, the more the ability to amplify, pull DNA from ancient samples improves. But again, the research continues.

– Does your method increase the number of DNA pieces?

– Yes, in fact, this mixture corrects breaks in DNA or oxidized nucleotides and allows you to work with these pieces when preparing samples for sequencing.

– Let's move on to another interesting area called synthetic biology. How rapidly is it developing and why did it appear?

– Synthetic biology is an umbrella name that covers a lot of different areas, starting with the same genomic editing and ending with the creation of modified organisms. For ourselves, we define synthetic biology as a kind of semi-engineering direction that uses the fundamental knowledge accumulated in molecular biology and biochemistry over the past fifty years since the discovery of the properties of DNA as a carrier of genetic information. In 2023, the community of geneticists and biochemists will celebrate the 70th anniversary of the discovery of how genetic information is stored in DNA.

Synthetic biology originated as an attempt to learn how to predict how specific systems will behave at the level of individual proteins, molecules and whole organisms on the basis of accumulated knowledge, and on the basis of this to create proteins and cells of body tissues with specified parameters and characteristics. Since this branch of science does not have a dedicated object of study, we have a wide disparate set of attempts to obtain modified biological systems of different levels.

There are "wow moments" from time to time. For example, a few years ago I liked a video where scientists forced human cells to produce spider web proteins. And they had a very spectacular demonstration. Scientists took a piece of tissue seeded with these cells, shot at it with a pistol, and the bullet did not penetrate it, because the web is extremely strong to break. They advertised the technology as a possible new synthetic fiber for body armor. But since then we haven't seen any new bulletproof vests. So far, this can be considered a spectacular experiment that shows what we can achieve in principle.

Work is underway with specially bred plants that begin to fluoresce if there is an explosive in the soil. In fact, synthetic biology is humanity's attempts to make biological objects more understandable for manipulation.

– Are there any applications, for example, for creating tissues or organs?

– Very active work is being done with tissues and organs now. In fact, it is one of the main directions of development, but so far we are limited to what we are trying to make tissues and organs that have not yet been modified. More recently, there has been a wave of expectations about stem cells. It seemed as if we could take any human cell and reprogram it into a stem cell, and then into some other. And in general, it is possible. But we still cannot create artificial tissues and organs with the cell density that would allow them to be used normally in medicine. Although the direction is certainly developing so actively that we will come to this soon. In Russia, such work is also underway.

– Where, for example?

– For example, in Skolkovo there is a company "3D Bioprinting Solutions", which is engaged in 3D bioprinting or the manufacture of synthetic matrices, which are then seeded with human cells, which can become the basis for obtaining tissues and organs in the future.

– Let's summarize a certain result of our conversation. Do you think manipulations with human genes are dangerous or for the common good?

– You understand that this question is completely rhetorical. There is not a single technology in the world that can be called 100% good or evil. Of course, like any technology, all biological technologies can be used for the benefit of humanity, and can carry significant risks. And, in fact, the task of scientists is to conduct a dialogue with society, to explain what opportunities science has, why some concerns are justified and some are unfounded. Only together can compromises be reached on the usefulness of a particular new technology. Of course, each of them will always involve some risks, but, in general, humanity has long learned to accept risks and somehow manage them.

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