vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-25-04vavilov-4473Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYИзучение конкатенации баркодированных трансгенов, линеаризованных Cas9Studying concatenation of the Cas9-cleaved transgenes using barcodeshttps://orcid.org/0000-0001-5152-9914СмирновA. В.SmirnovA. V.

Новосибирск

Novosibirsk

КораблевA. Н.KorablevA. N.

Новосибирск

Novosibirsk

СероваИ. А.SerovaI. A.

Новосибирск

Novosibirsk

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

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Novosibirsk

МуравьёваA. А.MuravyovaA. A.

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Novosibirsk

ВалеевE. С.ValeevE. S.

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Novosibirsk

https://orcid.org/0000-0003-0611-0203БаттулинН. Р.BattulinN. R.

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Novosibirsk

battulin@gmail.comФедеральный 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 FederationНовосибирский национальный исследовательский государственный университетРоссияNovosibirsk State UniversityRussian FederationФедеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук; Новосибирский национальный исследовательский государственный университетРоссияInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences; Novosibirsk State UniversityRussian Federation2025030320252912634Copyright © Смирнов A.В., Кораблев A.Н., Серова И.А., Юнусова A.М., Муравьёва A.А., Валеев E.С., Баттулин Н.Р., 20252025Смирнов A.В., Кораблев A.Н., Серова И.А., Юнусова A.М., Муравьёва A.А., Валеев E.С., Баттулин Н.Р.Smirnov A.V., Korablev A.N., Serova I.A., Yunusova A.M., Muravyova A.A., Valeev E.S., Battulin N.R.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/4473

   При пронуклеарной микроинъекции эндонуклеаза Cas9 используется для создания двуцепочечных разрывов ДНК in vivo в геномном целевом локусе или внутри донорского вектора для улучшения интеграции трансгена. Взаимодействие Cas9 с факторами репарации ДНК во время обработки концов трансгена и интеграции – актуальная тема. Ранее мы разработали генетическую систему на основе баркодов для анализа рекомбинации трансгенов после пронуклеарной микроинъекции у мышей. При этом подходе плазмидная библиотека линеаризуется ферментом рестрикции или комплексом Cas9:gRNA на участке между парой баркодов. Пул баркодированных молекул вводится внутрь пронуклеуса, что приводит к образованию многокопийных конкатемеров. В представленной статье мы сравнили эффекты разрезания Cas9 в условиях in vivo (эксперимент RNP+) или для трансгенов, линеаризованных in vitro и очищенных через агарозный гель (эксперимент RNP–). В эксперименте RNP+ было обнаружено два трансгенных однокопийных эмбриона. В эксперименте RNP– было идентифицировано шесть положительных эмбрионов, четыре из них имели низкокопийные конкатемеры. Анализ баркодов методом NGS показал, что 53 % баркодированных концов поменяли свои исходные пары баркодов, что является признаком гомологичной рекомбинации между концами трансгенов. Из 20 слияний «трансген-трансген» 11 не имели мутаций и, по-видимому, были получены путем повторного лигирования тупых концов, полученных с помощью Cas9. Большинство мутировавших соединений содержало асимметричные делеции 2–4 нуклеотидов из-за специфического тримминга концов Cas9. Эти данные указывают на то, что связанная с Cas9 ДНК создает препятствия для конкатенации. В то же время свободные концы ДНК соединяются таким же образом, как и концы, обработанные рестриктазами, но с характерной асимметрией. Будущие эксперименты с пронуклеарными микроинъекциями CRISPR/Cas помогут понять, как CRISPR-нуклеазы влияют на репарацию ДНК.

   In pronuclear microinjection, the Cas9 endonuclease is employed to introduce in vivo DNA double-strand breaks at the genomic target locus or within the donor vector, thereby enhancing transgene integration. The manner by which Cas9 interacts with DNA repair factors during transgene end processing and integration is a topic of considerable interest and debate. In a previous study, we developed a barcode-based genetic system for the analysis of transgene recombination following pronuclear microinjection in mice. In this approach, the plasmid library is linearized with a restriction enzyme or a Cas9 RNP complex at the site between a pair of barcodes. A pool of barcoded molecules is injected into the pronucleus, resulting in the generation of multicopy concatemers. In the present report, we compared the effects of in vivo Cas9 cleavage (RNP+ experiment) and in vitro production of Cas9-linearized transgenes (RNP– experiment) on concatenation. In the RNP+ experiment, two transgenic single-copy embryos were identified. In the RNP– experiment, six positive embryos were identified, four of which exhibited low-copy concatemers. Next-generation sequencing (NGS) analysis of the barcodes revealed that 53 % of the barcoded ends had switched their initial library pairs, indicating the involvement of the homologous recombination pathway. Out of the 20 transgene-transgene junctions examined, 11 exhibited no mutations and were presumably generated through re-ligation of Cas9-induced blunt ends. The majority of mutated junctions harbored asymmetrical deletions of 2–4 nucleotides, which were attributed to Cas9 end trimming. These findings suggest that Cas9-bound DNA may present obstacles to concatenation. Conversely, clean DNA ends were observed to be joined in a manner similar to restriction-digested ends, albeit with distinctive asymmetry. Future experiments utilizing in vivo CRISPR/Cas cleavage will facilitate a deeper understanding of how CRISPR-endonucleases influence DNA repair processes.

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