Newswise — The CRISPR-Cas9 system is adapted for genomic editing of many different organisms, but the existing approaches to genetic modification often are rather laborious and inefficient. In particular, such a problem existed in the case of methylotrophic yeasts, which are widely used for the production of proteins valuable in pharmacology and food industry. Scientists from Research Center of Biotechnology RAS with colleagues developed a set of plasmids that deliver CRISPR-Cas9 component genes into cells in the form of individual DNA molecules that are combined into a single genetic construct directly in yeast. This allowed to significantly simplify the genomic editing procedure and achieve high efficiency of CRISPR-Cas9 introduction into yeast cells. The results of the work were published in International Journal of Molecular Sciences. The study was performed as part of the project of The Federal Scientific and Technical program for the development of genetic technologies and supported by the project “Science and Universities”.
Yeasts have long been used by humans, for example in wine making and bakery. They have also become one of the most important objects of biotechnology. In particular methylotrophic yeasts of the genera Ogataea and Komagataella have been in great demand for the production of proteins used in pharmacology and the food industry. It is possible to make yeast to produce molecules that are not peculiar to them, but necessary for humans, by "inserting" the gene of interest into the genome of these microscopic fungi. The CRISPR-Cas9 technology of genome editing, which allows to significantly restructure the metabolism of cells, offers even wider opportunities.
CRISPR-Cas9 is a system of bacterial immunity since it is used by microorganisms to "memorize" foreign DNA and RNA, such as those belonging to viruses that have ever tried to enter a cell. The principle of "memorization" is that Cas proteins, on the one hand, simply destroy foreign sequences to prevent infection, and, on the other, cut out fragments from the pathogen's genome, which are then embedded in a CRISPR cassette, simply put a collection of genetic regions of all those interveners encountered by the bacterium. Then RNA molecules are synthesized from the CRISPR cassette, which, together with the Cas9 protein, "patrol" the cell and look for familiar genetic sequences of "intruders".
Scientists have adapted the CRISPR-Cas9 system in order to insert with its help foreign, but often valuable from the biotechnological point of view genes not only into the bacterial cells, but also yeast, plants and even animal cells. In this way it is already possible to create disease-resistant crop varieties and improved animal breeds, as well as to treat (yet not massively) severe human diseases, such as sickle cell disease and β-thalassemia. However, existing genome editing approaches, including those for some yeast species, are rather laborious and inconvenient to use.
Scientists from the Research Center of Biotechnology RAS (Moscow) together with colleagues from Kurchatov Institute (Moscow) have proposed an approach that will significantly simplify the process of genome editing using the CRISPR-Cas9 system in methylotrophic yeast. Authors took as a basis two artificial plasmids - circular DNA molecules - one of which carried a gene coding for the Cas9 protein, and the second - a short RNA sequence recognizing the place in the genome that needs to be edited. In addition, the plasmids carried a gene conferring resistance to the antibiotic geneticin. This gene served as a marker, with the help of which it was possible to select those yeast cells in which the introduction of the plasmids was successful. Thus, if yeast were able to grow on the antibiotic-containing medium, it means that the CRISPR-Cas9 system was also present in them.
Researchers simultaneously introduced into yeast cells two plasmids described above, which were cleaved in a special way, so that they were able to "find" each other in the cell and connect the terminal parts into a single sequence. The resulting single plasmid was maintained in yeast cells autonomously, that is without introduction into the chromosome. After the CRISPR-Cas9 system delivered in this way had performed its gene editing function, the plasmid was lost by the cells. The authors demonstrated the effectiveness of the proposed approach by successfully editing the genomes of four species of methylotrophic yeast: Ogataea polymorpha, O. parapolymorpha, O. haglerorum, and Komagataella phaffii.
"Potentially our system can be used to modify multiple genes simultaneously, however, it has to be proved in the course of new experiments. At the same time, we already have a good tool for editing the genomes of methylotrophic yeasts, which expands the opportunities of their usage to create strains producing recombinant proteins, as well as application of metabolic engineering methods to them," says one of the authors of the study, Michael Agaphonov, Ph.D., head of the group of Genetic engineering of lower eukaryotes of Research Center of Biotechnology.
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