vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-24-65vavilov-4286Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYВлияние ауксин-зависимой деградации когезина и конденсинов на репарацию двуцепочечных разрывов ДНК в эмбриональных стволовых клетках мышиEffects of the auxin-dependent degradation of the cohesin and condensin complexes on the repair of distant DNA double-strand breaks in mouse embryonic stem cellshttps://orcid.org/0000-0001-5152-9914СмирновА. В.SmirnovA. V.

Новосибирск

Novosibirsk

hldn89@gmail.comhttps://orcid.org/0000-0003-1751-7586РыжковаА. С.RyzhkovaA. S.

Новосибирск

Novosibirsk

https://orcid.org/0009-0002-2982-5076ЮнусоваА. М.YunusovaA. M.

Новосибирск

Novosibirsk

Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian Federation202408102024286583591Copyright © Смирнов А.В., Рыжкова А.С., Юнусова А.М., 20242024Смирнов А.В., Рыжкова А.С., Юнусова А.М.Smirnov A.V., Ryzhkova A.S., Yunusova A.M.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/4286

Семейство SMC-белков, включающее когезин и конденсины I/II, играет ключевую роль в формировании топологической структуры хромосом и косвенно влияет на широкий спектр клеточных процессов, в том числе и на репарацию двуцепочечных разрывов ДНК (DSB). Комплекс когезина регулирует репарацию DSB на нескольких уровнях, например, распространяя сигнал γH2AX и удерживая концы ДНК в непосредственной близости за счет экструзии петель возле разрыва. Когезин также скрепляет сестринские хроматиды во время фазы S/G2, что ограничивает потенциальную подвижность концов ДНК. По имеющимся данным, в фибробластах человека нокдаун когезина стимулирует образование геномных делеций между удаленными DSB (3.2 тыс. п.о.), но не влияет на репарацию одиночных или близких DSB (34 п.о.). Мы решили проверить это наблюдение на эмбриональных стволовых клетках мыши, несущих ауксин-индуцибельный дегрон Rad21 (субъединица когезина) или Smc2 (субъединица конденсинов I+II). Для этого мы использовали нуклеофекцию RNP Cas9 и пары гайдовых РНК для генерации делеций и инверсий с высокой эффективностью без дополнительной селекции. Мы определили оптимальные условия для эффективной электропорации, включая настройки Neon, а также тайминги появления делеций. Были протестированы две стратегии добавления ауксина (суммарно четыре независимых эксперимента). Были исследованы частоты перестроек в двух сайтах размером около 3.5 и 3.9 тыс. п.о. Вопреки ожиданиям, деплеция Rad21 не увеличивала частоту делеций/инверсий, даже для региона с активной границей Ctcf. Фактически наблюдалось снижение частоты делеций (но не инверсий) на 12 %. Деплеция Smc2 не приводила к заметному увеличению частот делеций/инверсий, возможно, из-за высокой биологической изменчивости между экспериментами. Анализ TIDE показал, что частота редактирования была постоянной для большинства экспериментов (30–50 %), с незначительным снижением после добавления ауксина. В статье также обсуждается применимость метода Neon/ddPCR для создания и детекции делеций в эмбриональных стволовых клетках мыши.

The SMC protein family, including cohesin and condensin I/II, plays a pivotal role in maintaining the topological structure of chromosomes and influences many cellular processes, notably the repair of double-stranded DNA breaks (DSBs). The cohesin complex impacts DSB repair by spreading γH2AX signal and containing DNA ends in close proximity by loop extrusion. Cohesin supports DNA stability by sister chromatid cohesion during the S/G2 phase, which limits DNA end mobility. Cohesin knockdown was recently shown to stimulate frequencies of genomic deletions produced by distant paired DSBs, but does not affect DNA repair of a single or close DSBs. We examined how auxin-inducible protein degradation of Rad21 (cohesin) or Smc2 (condensins I+II) changes the frequencies of rearrangements between paired distant DSBs in mouse embryonic stem cells (mESCs). We used Cas9 RNP nucleofection to generate deletions and inversions with high efficiency without additional selection. We determined optimal Neon settings and deletion appearance timings. Two strategies for auxin addition were tested (4 independent experiments in total). We examined deletion/inversion frequencies for two regions spanning 3.5 and 3.9 kbp in size. Contrary to expectations, in our setting, Rad21 depletion did not increase deletion/inversion frequencies, not even for the region with an active Ctcf boundary. We actually observed a 12 % decrease in deletions (but not inversions). At the same time, double condensin depletion (Smc2 degron line) demonstrated high biological variability between experiments, complicating the analysis, and requires additional examination in the future. TIDE analysis revealed that editing frequency was consistent (30–50 %) for most experiments with a minor decrease after auxin addition. In the end, we discuss the Neon/ddPCR method for deletion generation and detection in mESCs.

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