CRISPR/Cas9 is looking for mutants
Russian scientists have developed a new method of rapid selection of cells with gene modifications using CRISPR/Cas9
Polina Kirsanova, ITEB RAS Press Service
A real revolution in genetic engineering was made by the discovery of the CRISPR/Cas9 system. CRISPR are short repeating regions in DNA (literally "grouped short palindromic repeats"), which together with Cas proteins (literally "CRISPR–related proteins") represent a kind of analogue of immunity in bacteria, they help microorganisms to protect themselves from viruses. CRISPR stores information about viruses that previously attacked bacteria, and Cas cuts the viral DNA for further destruction. Since 2013, the world scientific community has begun to use this system to edit the genes of not only bacteria, but also higher organisms (the first experiments on human cells are actively conducted), which opens up new opportunities for the treatment of all kinds of human diseases, including the most severe viral, oncological and hereditary (for example, HIV, Down syndrome, etc.).
Russian scientists from 6 major scientific organizations in Moscow and Pushchino have jointly developed a new strategy for obtaining cellular knockouts, which eliminates the long and painstaking procedure of growing and screening clones in order to find the desired mutation. The results of this major study can be found in more detail in the February issue of the journal Scientific Reports (Zotova et al., Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags).
The main developer of this approach, Dmitry V. Mazurov, Candidate of Medical Sciences, Head of the Cell and Gene Technologies Group at the Institute of Gene Biology of the Russian Academy of Sciences, shared with the press service of the ITEB RAS the main idea of the method "The new method we have developed for selecting edited cells is that cells with the desired gene modification exhibit a short peptide on their surface. With the help of an antibody to this peptide and using the method of cell sorting, it is possible to easily and quickly isolate the desired knockout cells by any genes, including products that are inside the cell or secreted into the external environment. The genetic construct that we have developed, which allows the peptide to be transported to the cell surface and simultaneously turn off the gene, is the shortest, but at the same time effective among the currently existing selection markers. We called our method SORTS (surface oligopeptide knock-in for rapid target selection), the abbreviation of which in translation is close to the word "sorting" and reflects the essence of the new method. SORTS turned out to be useful not only for irreversible inactivation of the gene, but also for the gene shutdown regulated by the so-called degron at the protein level, which makes it possible to study the function of vital genes."
The co-author of this work, Ehson Lukmanovich Kholmukhamedov, Doctor of Biological Sciences, Chief Researcher of the Laboratory of Pharmacological Regulation of Cell Resistance of the Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, confirmed the novelty and experimental advantages of the new approach: "The developed method provides fast and efficient selection of the obtained knockouts without a long and time-consuming method of cell cloning, which allowed us to quickly and reliably obtain three lines of genetically edited human embryonic kidney cells, knockout in the content of mitochondrial porins." The use of this method, according to the authors, "is suitable for any genes, and may also have additional advantages over existing methods of molecular biology." Using the example of human cell cultures infected with HIV, the team of authors has already demonstrated the possibility of destroying this virus, whose DNA is stably embedded in the human genome and cannot be removed by modern means of treatment. Work in this direction continues.
The research was supported by grants 18-14-00333, 17-14-01107 of the Russian Science Foundation, the program of fundamental scientific research of State Academies for 2013-2020, grants 18-29-07052, 18-04-01016 and 18-34-00712 from the Russian Foundation for Basic Research, as well as the project 14.Z50. 31.
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