vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ16.175vavilov-700Research ArticleАктуальные технологии генетики и селекции. ОБЗОРCurrent technologies in genetics and breeding. REVIEWСистема CRISPR/Cas9 – универсальный инструмент геномной инженерииCRISPR/Cas9, a universal tool for genomic engineeringСмирновА. В.SmirnovA. V.

Новосибирск

Novosibirsk

ЮнусоваА. М.YunusovaA. M.

Новосибирск

Novosibirsk

ЛукьянчиковаВ. А.LukyanchikovaV. A.

Новосибирск

Novosibirsk

БаттулинН. Р.BattulinN. R.

Новосибирск

Novosibirsk

battulin@gmail.comФедеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук»РоссияInstitute of Cytology and Genetics SB RASRussian FederationФедеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук»; Федеральное государственное автономное образовательное учреждение высшего образования «Новосибирский национальный исследовательский государственный университет»РоссияInstitute of Cytology and Genetics SB RAS; Novosibirsk State UniversityRussian Federation201626092016204493510Copyright © Смирнов А.В., Юнусова А.М., Лукьянчикова В.А., Баттулин Н.Р., 20162016Смирнов А.В., Юнусова А.М., Лукьянчикова В.А., Баттулин Н.Р.Smirnov A.V., Yunusova A.M., Lukyanchikova V.A., Battulin N.R.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/700

Система CRISPR/Cas9 была изначально описана как механизм адаптивного иммунитета архей и бактерий, однако после ряда модификаций она нашла активное применение в генной инженерии, благодаря способности вносить направленный ДНК-разрыв с помощью короткого программируемого 20-нуклеотидного района в направляющей молекуле РНК (single guide RNA, sgРНК). Обзор посвящен современным приложениям системы CRISPR/Cas9 в генной инженерии. В первой его части описан основной механизм действия CRISPR/Cas9, особо уделено внимание причинам неспецифической активности Cas9 (off-targets). Она выражается в связывании комплексом Cas9-sgРНК нецелевых геномных участков, имеющих лишь частичную гомологию с sgРНК, что может приводить к нежелательным мутациям в геноме. В обзоре обсуждаются недавние улучшения специфичности связывания Cas9 и подходов по расширению функций CRISPR/Cas9 для трансгенеза. Популярность системы CRISPR/Cas9 в основном обусловлена ее выдающимся потенциалом для генной терапии и геномной инженерии, и последние достижения в этих областях представлены в нашем обзоре. В частности, CRISPR/Cas9 была недавно использована для контроля заражения клеток ВИЧ (вирусом иммунодефицита человека) и исправления генетических нарушений, таких как мышечная дистрофия Дюшенна и пигментный ретинит, на культурах клеток и животных моделях. Программируемость CRISPR/Cas9 облегчает создание трансгенных организмов с направленными генными мутациями, встройками генов и крупными хромосомными перестройками. Система CRISPR/Cas9 оказалась особенно востребованной для пронуклеарной микроинъекции при получении трансгенных сельскохозяйственных животных в биотехнологии. Один из разделов обзора посвящен генетическим скринингам на основе CRISPR/Cas9, которые приводят к высокоэффективной идентификации новых генов и генных сетей во многих биологических процессах. Наконец, в обзоре рассматривается технология генных драйверов, основанная на СRISPR/Cas9, которая представляет собой мощный инструмент для модификации экосистем в обозримом будущем.

The CRISPR/Cas9 system was initially described as an element of archeal and bacterial immunity, but gained much attention recently for its outstanding ability to be programmed to target any genomic loci through a short 20-nucleotide sgRNA region. Here we review some modern applications of the CRISPR/Cas9 system. First, we describe the basic mechanism of the CRISPR/Cas9 DNA recognition and binding, focusing in particular on its off-target activity. The CRISPR/Cas9 off-target activity refers to a non-specific recognition of genomic sites that have partial homology with sgRNA, occasionally resulting in unwanted mutations throughout the genome. We also note some recent improvements for enhancing Cas9 specificity or adding new functions to the system. Since Cas9-related hype is mostly driven by its remarkable potential for gene therapy and genome engineering, the latest CRISPR/Cas9 applications in these areas are also covered in our review. For instance, the CRISPR/Cas9 was recently used to control HIV infection and to repair genetic abnormalities, such as Duchenne muscular dystrophy or retinitis pigmentosa, both in cell cultures and rodent models. A programmable nature of CRISPR/Cas9 facilitates the creation of transgenic organisms through sitespecific gene mutations, knock-ins or large chromosomal rearrangements (deletions, inversions and duplications). CRISPR/Cas9 proved to be especially useful in pronuclear microinjections of farm animals as well, having strong impact on biotechnology. In addition, we review Cas9-augmented genetic screens that allow an unbiased search for new genes and pathways involved in a plethora of biological aspects, owing to Cas9 efficiency and versatility. Finally, we argue that gene drivers based on CRISPR/ Cas9 represent a powerful tool to modify ecosystems in the nearest future.

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