References

vavilov

Вавиловский журнал генетики и селекции

Vavilov Journal of Genetics and Breeding

2500-3259

Institute of Cytology and Genetics of Siberian Branch of the RAS

10.18699/VJ18.446

vavilov-1805

Research Article

КЛЕТОЧНАЯ БИОЛОГИЯ

CELL BIOLOGY

Способы повышения эффективности knock-in в геном плюрипотентных клеток человека при помощи системы CRISPR/Cas9

Improvement of the knock-in effciency in the genome of human induced pluripotent stem cells using the CRISPR/Cas9 system

https://orcid.org/0000-0002-7972-5949

Гридина

М. М.

Gridina

М. М.

gridinam@gmail.com

Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics, SB RASRussian Federation

2018

01012019

22810261032

2019

Гридина М.М.

Gridina М.М.

This work is licensed under a Creative Commons Attribution 4.0 License.

https://vavilov.elpub.ru/jour/article/view/1805

Индуцированные плюрипотентные стволовые клетки человека (чИПСК) – мощный инструмент для биомедицинских исследований. Возможность создания пациент-специфичных плюрипотентных клеток и последующая их дифференцировка в любой тип соматических клеток делают чИПСК замечательным объектом для создания in vitro моделей заболеваний, скрининга лекарственных препаратов и будущим источником клеточного материала для регенеративной медицины. Для того чтобы потенциал технологии чИПСК можно было реализовать в полном объеме, необходимы эффективные и точные методы редактирования генома этих клеток. В настоящее время система CRISPR/Cas9 – наиболее широко используемый подход для введения в ДНК сайт-специфичных двуцепочечных разрывов. С ее помощью с высокой эффективностью удается реализовать knock-out интересующих исследователя генов. Однако введение в целевое место генома заданной последовательности (knock-in) является существенно более сложной задачей. В зависимости от выбранного для проведения встройки локуса эффективность knock-in в геном чИПСК может составлять от 1 × 10–5 до 1 × 10–6, что на порядок ниже, чем показано для эмбриональных стволовых клеток мышей или перевивных линий клеток. В этом обзоре я делаю попытку объединить и структурировать всю известную информацию, касающуюся увеличения эффективности получения целевых встроек в геном чИПСК. В статье перечислены наиболее эффективные стратегии разработки донора для гомологичной рекомбинации, способы управления путями восстановления двуцепочечных разрывов, внесенных нуклеазой, в том числе за счет управления временем работы системы CRISPR/Cas9 в клетке. Низкая выживаемость чИПСК в результате проведения экспериментов по редактированию генома – еще одно затруднение на пути к успешному получению knock-in, для устранения которого предложено несколько высокоэффективных подходов. Наконец, я описываю, на мой взгляд, наиболее многообещающую стратегию получения линий чИПСК с целевой встройкой, которой является одновременное проведение редактирования и репрограммирования генома.

Human induced pluripotent stem (hiPS) cells are a powerful tool for biomedical research. The ability to create patient-specifc pluripotent cells and their subsequent diﬀerentiation into any somatic cell type makes hiPS cells a valuable object for creating in vitro models of human diseases, screening drugs and a future source of cells for regenerative medicine. To realize entirely a potential of hiPScells, eﬀective and precise methods for their genome editing are needed. The CRISPR/Cas9 system is the most widely used method for introducing site-specifc double-stranded breaks into DNA. It allows genes of interest to be knocked out with high efciency. However, knock-in into the target site of the genome is a much more difcult task. Moreover, many researchers have noted a low efciency of introducing target constructs into the hiPS cells’ genome. In this review, I attempt to describe the currently known information regarding the matter of increasing efciency of targeted insertions into hiPS cells’ genome. Here I will describe the most eﬀective strategies for designing the donor template for homology-directed repair, methods to manipulate the double-strand break repair pathways introduced by a nuclease, including control of CRISPR/Cas9 delivery time. A low survival rate of hiPS cells following genome editing experiments is another difculty on the way towards successful knock-in, and here several highly eﬀective approaches addressing it are proposed. Finally, I describe the most promising strategies, one-step reprogramming and genome editing, which allows gene-modifed integration-free hiPS cells to be efciently generated directly from somatic cells.

индуцированные плюрипотентные стволовые клетки человекасистема CRISPR/Cas9редактирование геномаэффективность knock-in

human induced pluripotent stem cellsCRISPR/Cas9 systemgenome editingknock-in efciency

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