Genetic affairs of the craftswomen

The history of Nobel works is not only the history of discoveries, but also the history of human destinies, the drama of ideas and a kaleidoscope of characters. The recent award of Nobel Prizes in chemistry was no exception. On October 7, the Nobel Committee called the names of the laureates of the Chemistry Prize. They were Emmanuel Charpentier from France and Jennifer Anne Doudna from the United States. The official wording of the basis for awarding the award: "For the study of the genome editing method." It is impossible to call such a solution in any way, the scientific community, and the winners themselves were waiting for this event a year ago. This is easily visible in a series of interviews, which the heroines of 2020 confidently gave back in 2018-2019, as if already overshadowed by the glory of the most authoritative scientific award. A feature of the 2020 bonus was a recordly short temporary interval from the first publications of laureates dedicated to the genome editing system to the very event itself. Many immediately started talking about the triumph of female science, but here I would not want to touch gender issues, but simply take a closer look at the personality of the heroines and the background of research.

Emmanuel Charpentier herself recalls how at the age of 12 she announced to her mother that she would work at the Pasteur Institute. Its scientific biography began in 1993 with the study of antibiotic resistance of microorganisms pathogenic for humans. This problem has become one of the main challenges of modern science about infectious diseases, and it is not surprising that the young microbiologist has chosen this particular direction for herself. She stubbornly analyzed hundreds of clinical insulators for their resistance to tetracycline, identified genetic material, and manually determined the sequences of the genes responsible for drug stability. The volume and nature of this work can be evaluated by anyone who is familiar with classical microbiology and molecular biology - tens of thousands of Petri's crossed cups, many hours in front of a gas burner.

The first work of Emmanuel was released in 1993 in the Gene magazine and was devoted to the characterization of resistance genes to tetracycline - a solid standard study, another bar to the general picture of genetic variability and adaptation of bacteria. After receiving a degree in 1995, Emmanuel remained true to the problem of resistance to antibiotics and continued to work in the United States. She chose scientific tasks, and ended up in a children's research hospital in Memphis in the Department of Infectious Diseases. And the first noticeable success is the publication in Nature in 1999 on the role of a two -component signal system of streptococci in determining the resistance to the reserve antibiotic of Vancromicin, however, in the modest role of one of five co -authors. Then followed the cycle of work on the study of individual components of transport and metabolic systems of staphylococci, one way or another involved in pathogenicity and antibiotic resistance. And suddenly - an unexpected turn towards the role of small RNA non -dodging RNA in the physiology of bacteria. What is this - scientific intuition or conjuncture? Perhaps, both of them: interest in these molecules arose in the early 2000s, and for the first time they started talking about them as regulatory back in 1967, which Emmanuel did not fail to mention in her review, published in 2007. This is a typical example of a revival in a new level of interest in the old and, it would seem, beyond the unnecessarily covered with dust “strange” results. But so far the interest of the future Nobel laureate remained only theoretical - another review followed in 2010. And no hint of CRISPR-CAS!

And suddenly a long -standing love for the systems of pathogenicity of bacteria, their ability to interspecific migration in bacterial populations miraculously “slammed” with interest in the tempting world of small RNAs and their biogenesis. This crossing of scientific interests “shot” in the form of an article in Nature in 2011. In it, one of the cutting mechanisms of the very guides of the RNA guides that together in enzymes-endonucleases is the “salt” of the genetic immunity of CRISPR-CAS. This mechanism assumed the participation of the seemingly useless ballast of small non -dodging RNA.

By the time of publication, Emmanuel Charpentier about CRISPR-CAS systems was already known. The abbreviation CRISPR itself (Clustered Regularly Interspaced Short Palindrome Repeats, or grouped periodic palindrome of short repetitions found in the genome) was introduced into circulation by employees Jansen with co -authors. They were the first to pay attention to the fact that, following the repetitions, the genes of enzymes are necessarily followed, which either change the structure of DNA, twisting or unwinding it, or are able to cut nucleic acids pointily. Scientists have long been attracted by repeated fragments of genomes, it would seem meaningless from the point of view of coding of genetic information about proteins, but clearly not accidentally fixed in certain positions during evolution. Opportunities for their study have grown markedly as new genomes, primarily bacterial, in the early 2000s.

The next iconic event on the path of awareness of the functions of sequences described by the Dutch sequences was the results of two groups from France and Spain, which found that pieces of genomes from evolutionarily remote organisms - bacterial viruses, or bacteriophages are found in CRISPRS. This observation intrigued the researchers: why does the bacterial genome catch and preserves foreign DNA? The results of this analysis were published almost simultaneously in 2005. It is noteworthy that for the first time in the history of the study of CRISPRS there are names of scientists of Russian origin Alexander Bolotin and Alexei Sorokin , who then worked at the National Institute of Agricultural Research of France in the Laboratory of Microorganism Genetics. It would seem that a strange fact that did not have at that time a rational explanation was seen by the researchers and was brought to the attention of the scientific community. It seems that it was a turning point in the trajectory of events related to the Nobel discovery. Gradually, the understanding of the role of these mysterious elements of bacterial genomes began to grow with facts extracted from the comparative analysis of genetic texts and from experiments. And further all this way, our compatriots who worked in Russia and abroad continued to make a considerable contribution to the progressive development of a new direction primarily in the form of new original ideas. In 2006, in the work of Kira Makarova, Svetlana Shabalina, Nikolai Grishin, Yuri Wolf and Evgeny Kunin, from national health institutions in the United States, it was first suggested that CRISPRS, along with the side of enzymes, are the ancient system of genetic immunity of bacteria, designed to limit the infection of bacteriophages. The conclusions of scientists were based on a serious analysis using bioinformation methods. It was really beautiful and, perhaps, even the ingenious work of foresight, but, alas, without visual experimental confirmation. The story is repeated (recall the Nobel Prize for opening a telomerase) not at all as a farce, but as a kind of pattern, when it is precisely the scientists of Russian scientific schools “on the tip of the pen” that open new mechanisms, express a practically established concept, which then picks up and receives confirmation.

And so happened in 2007, when a group of researchers from the largest US food industry Danisco Inc. I published an article where Rudolf Barrangu (Rodolphe Barrangou) was the first in the list of authors in Science magazine. A short article on three pages of the printing text narrated that the presence of Streptococcus Thermophilus CRISPR cassettes containing fragments of the bacteriophage genome leads to an increase in resistance to phagish infection. Scattered facts formed a holistic picture. Such a publication could not go unnoticed, especially since it was preceded by a short editorial commentary, emphasizing the general biological significance of this work. The fact that the study was conducted not at any large university, not at the national health institutions of the United States, but in the RD-division of a food company engaged in the development of new probiotics based on lactic acid bacteria, is noteworthy. Microbiologists have done the work, who to this day continue to distinguish and cross new strains of lactobacill and bifidobacteria for the benefit of healing of mankind. It is worth thinking about the advisability of developing scientific groups based on biotechnological companies. A clear understanding of the pressing problems of production and the urgent need to look for their solution based on the achievements of fundamental science sometimes become prerequisites for real scientific breakthroughs. Perhaps this is the secret of the unusually high concentration of the Nobel laureates in “individual camps”, which is not from fundamental science require a quick applied effect, but, on the contrary, in high -tech companies give the right to life to a small island of fundamental science?

Further events developed like a snowball, so far without the participation of the Nobel Stars of 2020. And again, our colleagues, now from Moscow, from the Gena Institute of Biology, Konstantin Severinov and his employees have been contributing, examining the mechanisms of expression of the genes of the CRISPR-CAS system and publishing the results in 2009-2010. During this period of early development of CRISPR-CAS research, the names of Russian scientists and immigrants from Russia make up a little less than a third in the total list of publications. How can we not think about what potential in the field of life sciences we had in the early 2000s and, I would like to hope, have not yet been lost?

Rudolf Barrangu was happy to borrow the metaphor of Evgeny Kunin (then their numerous joint publications and very productive cooperation followed) regarding the role of CRISPR-CAS as an immune system of bacteria, and the victorious procession of a new genetic mechanism in world science began. This really became a milestone in the development of genomics and all modern biology. A network of interdependent events in a cage developing in time requiring the participation of a whole complex of different in nature of biomolecules was reconstructed, and material carriers and the sequence of these events were identified.

But there was some kind of understatement. It seems to be clear how the system generally works, of which components it consists of. But modern biology is not only a description of the processes, but also an attempt to look deep into the mechanism of the phenomenon itself at the atomic-molecular level. It is known that there is a cut of alien DNA, that the cutting tool (the DNKA enzyme), in all likelihood, is delivered using the RNA guides guides. But what is happening inside the molecular complexes, how are their components come into contact with each other and is an enzymatic act itself? This was still to be found out. This required other methods from the field of structural biology and chemical enzymology and other people.

So this whirlpool of events was involved in the professor of the University of California in Berkeley, the chemist-enzimologist Jennifer Dudna. Her colleague, genomic specialist Jilian Benfield from the same university, working with many nucleotide sequences of bacteria and observing different combinations of CRISPR-CAS components in genomes, was convinced of the need to further study at the level of intermolecular interactions and structures. She looked at the work of her colleagues nearby and simply called Jennifer in a neighborly way to deal with these problems. The choice was not accidental, by this time Professor Dudna was considered an already established ribozima specialist and structural analysis of RNA, including those small non-dodging RNAs that make up one of the screws in the mechanisms of CRISPR-CAS. Here, the phenomenon of scientific communications and relationships in the scientific community was manifested, when in order to go beyond their own capabilities in solving a problem, it is necessary to take a step towards colleagues, share the idea and give rise to a new job. An alternative option was also possible - “not to get an idea from the hut”, try to do experiments on their own. A collaboration was chosen.

I must say that the path to the science of the second laureate also began with childhood. A family friend, a biologist, instructed a schoolgirl during the summer holidays to prepare painted drugs of microscopic shells. The girl was fascinated by how, during staining, the structures invisible in native organisms manifest and can be seen proteins interacting with calcium ions. The choice was made - chemistry in the application to biology. Jennifer, relying on her experience and skills, in a short time made a number of solid classical works on the structure of the CAS9 enzyme, related enzymes from other bacteria, protein complexes - DNA and protein - RNA. These results became available to the scientific community in 2009-2011. In the same period, a meeting took place with Emmanuel Charpentier, who poured into a joint work that appeared in the SCIENCE magazine in 2012. For the first time, the in vitro system was simulated a directed replacement for DNA and shows which components of the supramolecular complex play a decisive role in the accuracy and effectiveness of manipulations with DNA. Easy in its idea and elegant work, which initially had its goal to understand how a multicomponent molecular machine works. It was completed 50 by 50% by the employees of the groups of Jennifer and Emmanuel in Berkeley, Vienna and Minda (Shvetia). Thanks to these studies, it became obvious that a programmable tool for intervention in the genome appeared in humanity. Two girls were not at a loss and announced this method in the article in Nature on March 6, 2013 under the meaningful name "Biotechnology: Reducting the genome."

However, 36 days earlier, on January 29, four articles in Nature were published at once with a demonstration of directed editing the genome no longer in vitro , but on the culture of mammalian cells, bacteria and fish embryos. Two more articles were published on February 15 in the journal Science . Groups from Harvard, Rockefeller and Seoul Universities competed between themselves. The time was not just for the days, but on the clock. Probably, each of the scientists, understanding the meaning of his work, dreamed of a cherished medal. One can only guess what incredible tension and Stakhanov pace made it possible to be born of these results.

Over the past seven years, genomic editing technology irreversibly has become part of the tools of modern biological science. In our country, it is used for research purposes in leading institutions and scientific centers. It makes it possible, conducting targeted replacements in the genome of cultivated cells and in experimental animals, to understand the functions of vital genes, including those responsible for hereditary diseases. In the FIC “Pushchinsky Scientific Center for Biological Research of the Russian Academy of Sciences”, as part of the National Project “Science”, a laboratory of genomic editing and transgenesis was created, specializing in the directed modification of receptor proteins genes. So far, the first results were obtained on cell cultures, but researchers dream of getting transgenic experimental animals, which until recently were purchased only abroad. Undoubtedly, CRISPR-CAS technology is a tool, but it is the emergence of new methods that provides a qualitative leap in the development of science. After 2013, Jennifer Dudna and Emmanuel Charpentier continued to engage in the structure and physico-chemical mechanisms underlying the work of CRISPR-CAS. Similar systems from various microorganisms were discovered and investigated, and the details of the DNA-RNA-Belack complexes at the level of intra-molecular events were clarified, regulators that block the editing machine were characterized. It is quite natural that the awareness of unprecedented possibilities for interfering in the holy of the Holy of Living Organism, in its genome, which brings a new method, provoked a wide discussion of ethical problems related to its use in various layers of society. Future Nobel laureates did not stand aside from this discussion, speaking with software articles calling not to overestimate the possibilities of new technology and with caution extrapolate it for use in medicine, especially for manipulations with reproductive cells and embryos.

But what about one of the discoverers of the CRISPR-CAS system, which received experimental confirmation of its functionality, Rudolph Barrangwa? Он теперь профессор в Университете Северной Каролины, занимается пищевыми технологиями и поиском новых пробиотиков, основатель и главный редактор журнала CRISPR Journal , ставшего трибуной для продвижения в том числе и его собственных идей о перспективах этой технологии. Баррангоу остается фанатом CRISPR, микробиологом-экспериментатором, мечтающим с помощью геномного редактирования накормить человечество, а в будущем — излечить от моногенных наследственных заболеваний. На его машине вместо номера крупными буквами написано ”CRISPR”.

Перед Нобелевским комитетом стояла сложна задача. Ведь на пьедестале почета должны стоять в том числе и авторы вполне законченной теоретической концепции о роли CRISPR и первой доказательной работы — наш соотечественник Евгений Кунин и француз Рудольф Баррангоу. Далее экспериментаторы, вскрывшие внутреннюю механику работы молекулярной машины. И наконец целая команда ученых из Гарварда, Рокфеллеровского университета и из Сеула, воплотивших эти знания в жизнь и получивших первые организмы с отредактированным геномом. Комитет принял свое решение, возможно, по принципу симметрии…Так или иначе, присуждение премий в области науки несравнимо прозрачнее, нежели в случае премии по литературе и Нобелевской премии мира. Что бесспорно, так это то, что все участники этой истории — люди, для которых наука стала образом жизни, они способны прилагать колоссальные усилия при достижении цели, ловить на лету и воспринимать новые идеи. В этом отношении судьба Нобелевской премии по химии 2020 года являет нам неплохой урок, заставляющий задуматься о том, какие пока еще не осмысленные открытия варятся в котле сегодняшней физико-химической биологии, как не пропустить то самое жемчужное зерно. У российских ученых наравне с другими всё еще остается шанс занять верхнюю ступеньку пьедестала…

Ирина Масулис,
cand. Biol. наук, Институт биофизики клетки РАН ФИЦ
«Пущинский научный центр биологических исследований Российской академии наук»

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