Новые методы количественной оценки репарации двухцепочечных разрывов, основанные на CRISPR/Cas9

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В данном обзоре рассматриваются современные подходы к изучению репарации двухцепочечных разрывов ДНК (DSB) в клетках млекопитающих с использованием системы CRISPR/Cas9. Благодаря своей универсальности и эффективности эндонуклеаза Cas9 применяется во множестве генетических репортёров. Мы обсуждаем различные репортёры, основанные на флуоресценции, применяемые для мониторинга процесса восстановления. Также среди инновационных подходов на основе Cas9 особое внимание уделяется методам анализа как одиночных, так и множественных DSB, включая подходы DSB-TRIP и ddXR. Эти методы открывают новые возможности для исследования причин структурных перестроек или анализа любых геномных участков. Кроме того, в обзоре рассматривается, чем DSB, индуцированные Cas9, отличаются от DSB, создаваемых другими эндонуклеазами, и как эти особенности могут повлиять на механизмы восстановления ДНК. Понимание этих различий имеет решающее значение для планирования экспериментов, направленных на изучение репарации DSB.

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А. В. Смирнов

Институт цитологии и генетики СО РАН

Автор, ответственный за переписку.
Email: hldn89@gmail.com
Россия, Новосибирск

А. М. Юнусова

Институт цитологии и генетики СО РАН

Email: hldn89@gmail.com
Россия, Новосибирск

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2. Рис. 1. Флуоресцентные генетические репортёры DSB. а – Светофорный репортёр (Traffic light reporter). Вызванный Cas9, DSB репарируется или через HDR с использованием донорной последовательности GFP, или через NHEJ/MMEJ, которые приводят к сдвигу рамки считывания и активации mCherry [27]. б – Repair-seq применяет высокопроизводительный нокдаун генов репарации с помощью dCas9-KRAB и библиотеки гРНК CRISPRi. После интеграции вирусов в геном и нокдауна целевых генов популяция трансдуцированных клеток электропорируется рибонуклеопротеиновым комплексом (RNP) Cas9 и гРНК против сайта-мишени, находящегося рядом с гРНК CRISPRi. Это позволяет оценить вклад выключенного гена в репарацию DSB, проанализировав инделы Cas9 [25]. Цветами обозначены уникальные гРНК и экспрессирующие их клеточные клоны (не имеет отношения к сигналу флуоресценции)

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3. Рис. 2. Новые количественные репортёры DSB на основе CRISPR/Cas9. а – DSB-TRIP использует короткие последовательности транспозонов, встроенные в геном в случайных местах. Транспозоны содержат 16-нуклеотидный баркод, спаренный с сайтом-мишенью для Cas9, что позволяет просеквенировать баркод и инделы вместе (1) и ассоциировать их с различными хроматиновыми контекстами после локализации транспозона (2). Метод позволяет различать пути репарации, такие как NHEJ, MMEJ и репарацию, основанную на одноцепочечном шаблоне (SSTR), который имеет схожие начальные этапы с гомологичной рекомбинацией (HR) [19]. б – Метод ddXR объединяет индукцию делеций с помощью Cas9 и их обнаружение с ddPCR. ddXR позволяет определить частоты делеций и инверсий, используя один и тот же зонд [20]. в – Секвенирование конкатемеров основано на микроинъекции линеаризованной ДНК в пронуклеус, где ДНК остаётся связанной с Cas9 после разрезания (реакция с термически инактивированным белком используется в качестве контроля). Баркоды позволяют идентифицировать отдельные копии и исследовать пути репарации DSB путём секвенирования участков слияний между копиями

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4. Рис. 3. Особенности системы CRISPR/Cas9, которые могут повлиять на результат репарации DSB. Время связывания Cas9 и ДНК определяется неизвестными клеточными факторами. Сигнатуры инделов Cas9 зависят от характера концов ДНК после разрезания [66]. Белковые домены Cas9 влияют на репарацию DSB локально или позволяют переключать активность Cas9 в ходе клеточного цикла. Индуцибельные варианты Cas9 контролируют момент появления DSB с помощью фотоактивации (очень быстрый CRISPR) или химической стабилизации (DD-Cas9); DD – дестабилизирующий домен; PAM – мотив рядом с протоспейсером

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