References

vavilov

Вавиловский журнал генетики и селекции

Vavilov Journal of Genetics and Breeding

2500-3259

Institute of Cytology and Genetics of Siberian Branch of the RAS

10.18699/VJ20.643

vavilov-2715

Research Article

МОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯ

MOLECULAR AND CELL BIOLOGY

Изучение влияния детерминант митохондриального импорта в структуре нРНК на активность комплекса нРНК/SpCas9 in vitro

Study of the effect of the introduction of mitochondrial import determinants into the gRNA structure on the activity of the gRNA/SpCas9 complex in vitro

Закирова

Э. Г.

Zakirova

E. G.

Вяткин

Ю. В.

Vyatkin

Y. V.

Верещагина

Н. А.

Verechshagina

N. A.

Музыка

В. В.

Muzyka

V. V.

Мазунин

И. О.

Mazunin

I. O.

https://orcid.org/0000-0001-9718-9038

Орищенко

К. Е.

Orishchenko

K. E.

keor@bionet.nsc.ru

Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of Siberian Branch of the Russian Academy of SciencesRussian Federation

ООО «АкадемДжин»РоссияAcademGene Ltd.Russian Federation

Балтийский федеральный университет им. И. КантаРоссияImmanuel Kant Baltic Federal UniversityRussian Federation

Новосибирский национальный исследовательский государственный университетРоссияNovosibirsk State UniversityRussian Federation

Сколковский институт науки и технологийРоссияSkolkovo Institute of Science and TechnologyRussian Federation

2020

28082020

245512518

2020

Закирова Э.Г., Вяткин Ю.В., Верещагина Н.А., Музыка В.В., Мазунин И.О., Орищенко К.Е.

Zakirova E.G., Vyatkin Y.V., Verechshagina N.A., Muzyka V.V., Mazunin I.O., Orishchenko K.E.

This work is licensed under a Creative Commons Attribution 4.0 License.

https://vavilov.elpub.ru/jour/article/view/2715

О том, что нарушения структуры митохондриального генома приводят к широкому спектру нейромышечных и нейродегенеративных заболеваний, известно уже давно, но до сих пор не найдено эффективного метода лечения болезней митохондриального происхождения. В основном проблемы с терапией подобных заболеваний обусловлены состоянием гетероплазмии митохондриальной ДНК (мтДНК). Ввиду многокопийности митохондриального генома мутантные копии мтДНК часто сосуществуют с молекулами дикого типа в одной органелле. Клинические симптомы митохондриальных заболеваний и степень их манифестации напрямую зависят от количества мутантных молекул мтДНК в клетке. Смещая уровень гетероплазмии в сторону молекул дикого типа мтДНК, возможно добиться снижения негативного влияния мутации. Для этой цели разработано несколько генно-терапевтических подходов на основе TALE-нуклеаз и нуклеаз типа «цинковые пальцы», однако конструирование белковых доменов таких систем является долгим и трудоемким процессом. Cистема CRISPR/ Cas9 принципиально отличается от данных систем простотой использования, высокой эффективностью и механизмом действия. Все присущие характеристики и возможности системы делают ее перспективным инструментом в области генетической инженерии митохондрий. В настоящей статье мы впервые демонстрируем, что модификации направляющей РНК за счет вcтройки последовательностей, способствующих импорту нРНК в митохондрии, не влияют на функциональную активность комплекса нРНК/SpCas9 в условиях in vitro. Полученные результаты указывают на возможность модификации системы с сохранением ее функциональности и использования в перспективе для редактирования митохондриального генома.

It has long been known that defects in the structure of the mitochondrial genome can cause various neuromuscular and neurodegenerative diseases. Nevertheless, at present there is no effective method for treating mitochondrial diseases. The major problem with the treatment of such diseases is associated with mitochondrial DNA (mtDNA) heteroplasmy. It means that due to a high copy number of the mitochondrial genome, mutant copies of mtDNA coexist with wild-type molecules in the same organelle. The clinical symptoms of mitochondrial diseases and the degree of their manifestation directly depend on the number of mutant mtDNA molecules in the cell. The possible way to reduce adverse effects of the mutation is by shifting the level of heteroplasmy towards the wild-type mtDNA molecules. Using this idea, several gene therapeutic approaches based on TALE and ZF nucleases have been developed for this purpose. However, the construction of protein domains of such systems is rather long and laborious process. Meanwhile, the CRISPR/Cas9 system is fundamentally different from protein systems in that it is easy to use, highly efficiency and has a different mechanism of action. All the characteristics and capabilities of the CRISPR/Cas9 system make it a promising tool in mitochondrial genetic engineering. In this article, we demonstrate for the first time that the modification of gRNA by integration of specific mitochondrial import determinants in the gRNA scaffold does not affect the activity of the gRNA/Cas9 complex in vitro.

митохондриальная ДНКCRISPR/Cas9детерминанты импорта в митохондриигетероплазмия

mitochondrial DNACRISPR/Cas9the mitochondrial import determinantsheteroplasmy

This work was supported by state budget project of Institute of Cytology and Genetics SB RAS “Molecular genetic principles of regulation of gene expression, morphology, differentiation and reprogramming of cells” (No. 0324-2019-0042-C-01), by state budget project No. 0259- 2019-0001 and by the Russian Science Foundation No. 17-75-20015

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The authors declare that there are no conflicts of interest present.