medgenМедицинская генетикаMedical Genetics2073-7998Publishing House «Genius Media» LLC10.25557/2073-7998.2021.08.3-10medgen-1957Research ArticleОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯORIGINAL RESEARCHНокдаун NUDT16L1 (TIRR) повышает эффективность геномного редактированияNUDT16L1 (TIRR) knockdown increases genome editing efficacyАнучинаА. А.AnuchinaA. A.arinate@mail.ruСмирнихинаС. А.SmirnikhinaS. A.noemail@neicon.ruЗайнитдиноваМ. И.ZaynitdinovaM. I.noemail@neicon.ruДемченкоА. Г.DemchenkoA. G.noemail@neicon.ruМишинаО. С.MishinaO. S.noemail@neicon.ruЛавровА. В.LavrovA. V.noemail@neicon.ruФГБНУ «Медико-генетический научный центр имени академика Н.П. Бочкова»РоссияResearch Centre for Medical GeneticsRussian Federation202123112021208310Copyright © Анучина А.А., Смирнихина С.А., Зайнитдинова М.И., Демченко А.Г., Мишина О.С., Лавров А.В., 20212021Анучина А.А., Смирнихина С.А., Зайнитдинова М.И., Демченко А.Г., Мишина О.С., Лавров А.В.Anuchina A.A., Smirnikhina S.A., Zaynitdinova M.I., Demchenko A.G., Mishina O.S., Lavrov A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.medgen-journal.ru/jour/article/view/1957

Введение. Эффективная точная коррекция мутаций очень важна для внедрения CRISPR-Cas в качестве инструмента для потенциальной генной терапии. Механизм направленной гомологичной репарации (НГР), активируемый после внесения нуклеазой двунитевого разрыва, позволяет корректировать все существующие типы мутаций. Однако НГР не является доминирующим механизмом в клетке, что ограничивает ее эффективность. В нашем исследовании впервые описано, как фактор репарации NUDT16L1 может контролировать эффективность НГР при редактировании с помощью CRISPR-Cas. Цель: исследовать влияние нокдауна и гиперэкспрессии NUDT16L1 на эффективность НГР. Методы. Исследование проводили на клеточной культуре HEK293T. Систему CRISPR-Cas в плазмиде, а также вектор для гиперэкспрессии NUDT16L1 доставляли путём липофекции. Для нокдауна гена NUDT16L1 использовали малые интерферирующие РНК. Результаты. Нокдаун NUDT16L1 повышает уровень НГР в плазмидном и геномном локусах, что проявляется повышением доли GFP-позитивных клеток в 1,8-3,6 раз. Заключение. Нокдаун гена NUDT16L1 может быть использован для повышения эффективности исправления патогенных вариантов ДНК методом геномного редактирования.

Introduction. Effective precise knock-in is crucial for implementing CRISPR-Cas9 system as an efficient instrument for potential gene therapy. Homology directed repair (HDR) pathway allows correction of all types of existing mutations. However, HDR is not a major repair pathway of the cell that limits its efficiency. In our study, we present for the first time how repair factors NUDT16L1 controls HDR efficiency. Aim: to study an influence of NUDT16L1 knockdown and overexpression on the HDR efficacy. Methods. HEK293T culture was used to perform the research. Plasmid CRISPR-Cas system along with NUDT16L1 overexpression vector were delivered with lipofection. For NUDT16L1 knockdown small interfering RNAs were used. Results. We discovered that knockdown of NUDT16L1 enhances HDR both in the plasmid and genomic loci increasing eGFP signal from 1.8 to 3.6 times in HEK293T cells. Conclusion. NUDT16L1 knockdown could be used for enhancing of the pathogenic mutations correction through genome editing.

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