How to cut DNA
Siberian scientists are honing genomic editing tools
Olga Kolesova, "Search"
The emergence of "molecular scissors" – the CRISPR/Cas9 genomic editing system – has led to a rapid surge in genetic technologies. For the first time, a special CRISPR locus in bacteria was discovered by a group of Japanese scientists back in 1987. It took more than 20 years to develop an artificial CRISPR system supplemented with Cas proteins for targeted genome editing. Jennifer Doudna and Emmanuel Charpentier received the Nobel Prize in Chemistry in 2020 for the introduction of new methods of genetic engineering. However, as during any revolution, there are many issues behind the scenes that require close study.
"We are talking separately about DNA repair systems and genomic editing systems. But any genomic editing tools first damage the DNA. How do repair systems behave in this case, perfectly developed in the body? There is a "black box" between these two systems, about the events in which scientists do not know anything. In short, there were a lot of intriguing ideas for testing," says Dmitry Pyshny, Director of the Institute of Chemical Biology and Fundamental Medicine (IHBFM) of the SB RAS, Corresponding member of the RAS.
A grant from the Russian Science Foundation allowed the IHBFM SB RAS team to start real exploratory research that has no analogues in the world. The project "Modification of nucleic acids and DNA repair as a source of new genome management tools", in addition to scientific novelty, is characterized by an amazing diversity.
– The Institute has developed several scientific schools aimed at studying the processes of DNA repair. These are the schools of Olga Ivanovna Lavrik, Dmitry Olegovich Zharkov. Nikita Aleksandrovich Kuznetsov's group has excellent competencies in terms of physico-chemical analysis of protein-nucleic interactions," continues Dmitry Pyshny, project manager. – In addition to the interaction of repair systems and genomic editing, we decided to investigate what the modification of nucleic acids that help CRISPR/Cas recognize the desired gene will lead to. Although it is not very advertised, but geneticists know that the molecular machine that makes changes to the genome does not always work correctly. And the problem of increasing accuracy is one of the most painful. The balanced efforts of chemists and enzymologists (enzymology is the science of enzymes) can solve it by modifying RNA conductors, which are just responsible for recognizing the desired site. In addition, Nikita Kuznetsov's group plans to master the synthesis of rather long single-stranded nucleic acids to create new genetic constructs. At the same time, we want to try to build an editing system in a different way, perhaps to create new molecular machines capable of recognizing DNA sections and making directional changes to the structure of the genome. The grant is designed for 4 years, I think during this time we will be able to create some hybrid molecules for genomic editing, which carry blocks and domains of different natural objects.
"Zinc fingers" and "molecular scissors"
– Genetic technologies were born at the turn of the 1970s and 1980s, when biologists learned how to accurately move DNA fragments from one place to another. In parallel, thanks to the development of the chemistry of oligonucleotide synthesis, DNA sections of the necessary sequence began to be obtained," says Dmitry Zharkov, Corresponding member of the RAS, Head of the Laboratory of Genomic and Protein Engineering of the IHBFM SB RAS. – Now genetic technologies mean changing the genetic material directly in living cells. The first tools for this appeared back in the 1980s: proteins of the "zinc finger" class were discovered, capable of recognizing certain DNA sequences. Then scientists discovered TALE class proteins in bacteria that cause plant diseases, which also recognize the necessary sequences in the host genome and change the activity of genes. However, these tools were not very convenient for genomic engineering: for each sequence that we wanted to change, we had to synthesize a new protein. Therefore, articles that thanks to the new CRISPR/Cas system, it is possible to specifically recognize a section of DNA using a small RNA molecule and make breaks directly into the genome of human cells caused a furor among geneticists. A real scientific revolution has taken place: a universal small and simply synthesizable instrument has appeared. This can be considered the beginning of the modern boom of genetic technologies. With the help of directed RNA, we introduce a gap in the right place of DNA, in this gap you can either embed the required DNA fragments, or turn off some genes when cross-linking with a mutation.
Many examples of the system's application have appeared, starting with the sensational and ethically ambiguous work of Chinese scientist He Jiankui on editing the genome of twin girls at the embryo stage and ending with the creation of a breed of pigs resistant to African plague. But no matter what excellent reviews have been received about the new tool, there are still significant obstacles to its use in medicine. The fact is that all currently known genomic editing systems have a certain non-targeted activity. And if we can close our eyes to the fact that the newly bred variety of corn will carry some additional mutations other than those programmed, then it is absolutely unacceptable that such mutations occur in the DNA of patients during genomic therapy. And the only way to check today is deep genome sequencing, which is very expensive and not suitable for mass use. Scientists around the world are working in two directions today. Firstly, they are trying to modify the active module to give the CRISPR/Cas system new functions. Secondly, they try to improve the accuracy of the system and avoid changes in other DNA fragments. Approaches to this can be very different. In particular, there is clearly insufficient research on this: it is not the protein itself that needs to be changed, but the chemical structure of the nucleic acid that is associated with it. Perhaps by modifying nucleic acids, we will be able to increase the accuracy of genomic editing. Considering that our institute has the strongest nucleotide chemistry in Russia, it is not surprising that it was decided to try to do this within the framework of the RNF project.
With surgical precision
Classical methods of genetic engineering imply that the researcher uses a biomaterial containing genomic DNA or mRNA, and with the help of well-described procedures clones the target gene with which he works further. This is quite easy to do if we are talking about a person or a mouse. But what about exotic organisms or those that no longer exist?
– The problem of obtaining biological material containing the DNA of such an organism is extremely difficult, – says Nikita Kuznetsov, Doctor of Chemical Sciences, Head of the Laboratory of Genetic Technologies of the IHBFM SB RAS. – That is why our direction of the project synthesis of extended oligonucleotides is relevant for the purpose of their further application in new approaches to the creation of genes.
Today, oligonucleotides are synthesized by a chemical method that has limitations on the length of the synthesized fragment. The enzymatic method of synthesis of oligonucleotides, which we are developing, will significantly increase the length of synthetic DNA fragments. For this purpose, we use one of the human enzymes – terminal deoxyribonucleotidyltransferase, capable of attaching nucleotides to any available 3'-terminal DNA fragment. However, in the body, the addition of nucleotides occurs randomly. We plan to make the enzyme work so that the oligonucleotides are attached in a certain sequence set by the researcher. To do this, after each addition, the enzyme must be stopped using modified nucleoside triphosphates. At our institute, a modification of nucleoside triphosphate was proposed, which has never been used in the world for this type of reactions with enzymes. We tested this approach in the first year of work on the project. The chemical part of the promotion is very serious. But it was found that the natural enzyme turned out to be too "gentle" to work: it does not tolerate high temperatures well, it is quickly inactivated. Therefore, our task in the near future is connected with obtaining a highly active temperature-resistant enzyme. We use two complementary approaches. Firstly, by molecular modeling, we check dozens of variants of mutant forms of the human enzyme that may have the necessary properties. The variants selected in this way will be obtained by site-directed mutagenesis methods and then tested in real conditions. Secondly, we are looking for an enzyme in nature that is similar in properties, but resistant to high temperatures. For example, like bacteria living in geysers or hot springs. Active work is underway in this direction. By the way, this enzyme is used in some test systems aimed at studying the effects of various substances on DNA. And by launching the enzymatic synthesis of extended DNA fragments, we simultaneously contribute to the development of diagnostic systems.
– It's not for nothing that even the name of the project sounds "creating new tools", – emphasizes Dmitry Pyshny. – We see the main interest in the use of these tools for genomic editing, but they can be used in other areas. Like a screwdriver, figuratively speaking. Nucleic acid is a universal object of molecular biology. And the tools, methods, algorithms, enzymes that we have created should be widely used. When we talk about the biotechnological part of the project, it is necessary to study in detail the process of the enzyme itself in order to understand how it can be optimized.
– In 2021, thanks to the grant funds, we bought a device for studying fast-flowing enzymatic processes Quench-flow RQF–3 (KinTek Corp., USA) - it allows you to stop the enzymatic reaction at the right time, starting from 5 milliseconds after the start. A detailed study of the mechanism of action of the enzyme requires such a specific technique," adds Nikita Kuznetsov.
Artificial out of competition?
The introduction of any changes to the genome of a living organism is accompanied by the inclusion of DNA repair systems responsible for the stability, safety, integrity of genetic information. The project was based on a simple idea: to see in vitro and in vivo how the CRISPR/Cas genomic editing system competes with the repair systems available in the cell, which, by the way, work extremely efficiently.
– CRISPR/Cas is an artificial system, it introduces specific mutations into the genome. And the repair systems, which consist of ensembles of proteins, seem to interfere with her work. This hypothesis must be tested in order to avoid possible difficulties for the targeted effect of the CRISPR/Cas system," explains Academician Olga Lavrik, Head of the Laboratory of Bioorganic Chemistry of Enzymes of the IHBFM SB RAS. – In our laboratory, the corresponding repair proteins are being studied, which interact very effectively with DNA breaks in the cell, DNA ligases and poly–(ADP-ribose)- polymerases. During the first year of work on the project, we looked at the interaction of these "sensors" of breaks with the CRISPR/Cas system using biochemical experiments. Surprisingly, the first results show that the artificial system competes very successfully with the natural one.
Our explanation of these results: the CRISPR/Cas system for eukaryotic cells is not native, nature did not prepare them for interaction with it. I can draw an analogy with protein biosynthesis: the cell very accurately includes the required natural amino acid in the synthesized protein on the ribosome, replacement never occurs. But once an artificial synthetic analog of an amino acid is introduced into the biosynthesis system, this analog is included in the synthesized protein. The control mechanisms do not recognize an unknown "alien". Now we need to test this hypothesis in living cells. Moreover, we will conduct a comparative analysis: we have already made cells in which certain nic sensors - proteins that recognize a DNA break – have been artificially removed. Let's see if the work of the "molecular scissors" differs in the presence of repair proteins and in the absence of them. There is no reliable data on this yet, although the idea literally lies on the surface. Therefore, in order to stay at the forefront, it is necessary to work quickly and efficiently.
If we really prove that a cell does not recognize an artificial CRISPR/Cas system that works well with DNA, this will be an excellent conclusion in terms of the prospects for genomic editing. But it's too early to put an end to the research. Since there are RNA conductors in this system, it is necessary to study how various RNA-binding proteins affect genomic editing.
–The fact is that genomic editing depends on the RNA guides themselves, which determine its direction, so we need to create an algorithm for the targeted modification of these RNA guides to ensure the most reliable and accurate operation of molecular genomic editing machines," Dmitry Pyshny continues. – This is one of the tasks that have not yet been worked out in world science. And the support of the Russian Science Foundation is very timely here – we managed to attract funding to test exactly the ideas. Moreover, the ideas are interdisciplinary. As part of the state task, as a rule, more traditional areas are funded. But in order to keep up with world science, we need programs that allow us to create something for use in the real sector of the economy. During the implementation of the project, we expect to receive truly new tools for genomic engineering that will find application in medicine, biotechnology, and agriculture.
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