How to shift the equilibrium of dna break repair in favor of homology recombination

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Abstract

With the practical implementation of the CRISPR/Cas technology for targeted genome editing, it has become possible to carry out genetic engineering manipulations with eukaryotic genomes with high efficiency. One of the key stages of this technology is the targeted induction of site-specific DNA cleavages (breaks). The cell repairs these breaks via one of two pathways: nonhomologous end joining or homologous recombination. The choice of DNA repair pathway is determined by the architecture of the sites at the DNA break area formed as a result of terminal resection and depends on the phases of the cell cycle. Nonhomologous end joining is the main pathway for repair of double-stranded DNA breaks in mammalian cells. It involves a nonspecific ligation reaction, the accuracy of which depends on the structure of the ends of the break, and can result in various insertions or deletions in the target region of the genome. Integration of the desired sequence into the genome occurs along the path of homologous recombination, the implementation of which requires a matrix with homology regions on both sides of the double-strand break. The introduction of a genetic construct into a given location in the genome is an important, but currently complex and labor-intensive task. At the same time, for fundamental studies of gene function and the creation of animal models of human diseases, the choice of the repair pathway can be of fundamental importance. This review is an attempt to combine and structure all known information on approaches to increasing the efficiency of DNA repair involving homologous recombination. The article lists the most effective strategies to shift the balance towards homologous repair, such as the use of inhibitors of the non-homologous end joining mechanism, regulation of key factors of homologous recombination, control of the cell cycle, chromatin status, construction of templates for homologous recombination.

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About the authors

O. A. Averina

Institute of Functional Genomics, Lomonosov Moscow State University; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University; Department of Chemistry, Lomonosov Moscow State University

Email: svetlana@belozersky.msu.ru
Russian Federation, Moscow, 119991; Moscow, 119991; Moscow, 119991

S. A. Kuznetsova

Institute of Functional Genomics, Lomonosov Moscow State University

Author for correspondence.
Email: svetlana@belozersky.msu.ru
Russian Federation, Moscow, 119991

O. A. Permyakov

Institute of Functional Genomics, Lomonosov Moscow State University; Department of Chemistry, Lomonosov Moscow State University

Email: svetlana@belozersky.msu.ru
Russian Federation, Moscow, 119991; Moscow, 119991

P. V. Sergiev

Institute of Functional Genomics, Lomonosov Moscow State University; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University; Department of Chemistry, Lomonosov Moscow State University

Email: svetlana@belozersky.msu.ru
Russian Federation, Moscow, 119991; Moscow, 119991; Moscow, 119991

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Supplementary files

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2. Fig. 1. Variants of guide RNA.

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3. Fig. 2. Genome editing technologies ZFN, TALEN, CRISPR/Cas. ZFN is a site-specific DNA cleavage by a chimeric ZF nuclease constructed on the basis of a zinc finger DNA-binding domain (30 aa interacting with a triplet of nucleotides in DNA) and the catalytic domain of the FokI restriction endonuclease. TALEN is DNA cleavage by a chimeric nuclease TALE constructed by attaching the catalytic domain of the FokI endonuclease to DNA-recognizing TALE proteins; one monomer of the TALEN DNA-binding domain (34 aa) recognizes one nucleotide of the target DNA sequence. CRISPR/Cas – recognition of the target DNA sequence occurs with the help of gRNA, consisting of guide and trans-activating RNA (or with the help of egRNA, in which guide and trans-activating RNA are connected by a linker), and cleavage is carried out by Cas nuclease. PAM is a short NGG motif required for Cas binding and introducing breaks into DNA. DNA repair occurs in one of two ways: non-homologous end joining (NHEJ) or homologous recombination (HDR) with the participation of a donor DNA template. As a result of DNA break repair, a specific gene is knocked out (Knockout) or the target genetic construct is integrated into the genome (Knock-in). tr.ac. crRNA ‒ trans-activating crRNA.

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4. Fig. 3. Organization of the CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13 systems. Arrows indicate the sites of single-stranded DNA breaks by the catalytic domains of RuvC and HNH (Cas9 and Cas12) and the sites of RNA breaks by the HEPN protein (Cas13).

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5. Fig. 4. DNA break repair. The repair pathways HDR, NHEJ, MMEJ, which involve protein factors and nucleic acid metabolism enzymes (shown next to the arrows indicating the direction of the repair pathway), and the corresponding donor DNA matrices are shown.

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6. Fig. 5. Scheme of activation of the 53BP1-PTIP-RIF1-Sheldin-CST cascade. The arrows indicate the sequence of activation stages, and the rectangular inserts provide their description. The top right shows information on the role of the BRCA1 protein, involved in the HDR process, at different phases of the cell cycle.

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7. Fig. 6. Schemes of homologous repair (HDR) of dsDNA using dsDNA as donor templates.

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Согласие на обработку персональных данных с помощью сервиса «Яндекс.Метрика»

1. Я (далее – «Пользователь» или «Субъект персональных данных»), осуществляя использование сайта https://journals.rcsi.science/ (далее – «Сайт»), подтверждая свою полную дееспособность даю согласие на обработку персональных данных с использованием средств автоматизации Оператору - федеральному государственному бюджетному учреждению «Российский центр научной информации» (РЦНИ), далее – «Оператор», расположенному по адресу: 119991, г. Москва, Ленинский просп., д.32А, со следующими условиями.

2. Категории обрабатываемых данных: файлы «cookies» (куки-файлы). Файлы «cookie» – это небольшой текстовый файл, который веб-сервер может хранить в браузере Пользователя. Данные файлы веб-сервер загружает на устройство Пользователя при посещении им Сайта. При каждом следующем посещении Пользователем Сайта «cookie» файлы отправляются на Сайт Оператора. Данные файлы позволяют Сайту распознавать устройство Пользователя. Содержимое такого файла может как относиться, так и не относиться к персональным данным, в зависимости от того, содержит ли такой файл персональные данные или содержит обезличенные технические данные.

3. Цель обработки персональных данных: анализ пользовательской активности с помощью сервиса «Яндекс.Метрика».

4. Категории субъектов персональных данных: все Пользователи Сайта, которые дали согласие на обработку файлов «cookie».

5. Способы обработки: сбор, запись, систематизация, накопление, хранение, уточнение (обновление, изменение), извлечение, использование, передача (доступ, предоставление), блокирование, удаление, уничтожение персональных данных.

6. Срок обработки и хранения: до получения от Субъекта персональных данных требования о прекращении обработки/отзыва согласия.

7. Способ отзыва: заявление об отзыве в письменном виде путём его направления на адрес электронной почты Оператора: info@rcsi.science или путем письменного обращения по юридическому адресу: 119991, г. Москва, Ленинский просп., д.32А

8. Субъект персональных данных вправе запретить своему оборудованию прием этих данных или ограничить прием этих данных. При отказе от получения таких данных или при ограничении приема данных некоторые функции Сайта могут работать некорректно. Субъект персональных данных обязуется сам настроить свое оборудование таким способом, чтобы оно обеспечивало адекватный его желаниям режим работы и уровень защиты данных файлов «cookie», Оператор не предоставляет технологических и правовых консультаций на темы подобного характера.

9. Порядок уничтожения персональных данных при достижении цели их обработки или при наступлении иных законных оснований определяется Оператором в соответствии с законодательством Российской Федерации.

10. Я согласен/согласна квалифицировать в качестве своей простой электронной подписи под настоящим Согласием и под Политикой обработки персональных данных выполнение мною следующего действия на сайте: https://journals.rcsi.science/ нажатие мною на интерфейсе с текстом: «Сайт использует сервис «Яндекс.Метрика» (который использует файлы «cookie») на элемент с текстом «Принять и продолжить».