J 2017

An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System

BOHAČIAKOVÁ, Dáša, Tereza RENZOVÁ, Veronika FEDOROVÁ, Martin BARÁK, Michaela BOSÁKOVÁ et. al.

Basic information

Original name

An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System

Authors

BOHAČIAKOVÁ, Dáša (703 Slovakia, belonging to the institution), Tereza RENZOVÁ (203 Czech Republic, belonging to the institution), Veronika FEDOROVÁ (203 Czech Republic, belonging to the institution), Martin BARÁK (203 Czech Republic, belonging to the institution), Michaela BOSÁKOVÁ (203 Czech Republic, belonging to the institution), Aleš HAMPL (203 Czech Republic, belonging to the institution) and Lukáš ČAJÁNEK (203 Czech Republic, belonging to the institution)

Edition

Stem Cells and Development, New York, Mary Ann Liebert, Inc. 2017, 1547-3287

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10601 Cell biology

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 3.315

RIV identification code

RIV/00216224:14110/17:00095168

Organization unit

Faculty of Medicine

DOI

UT WoS

000413639900001

Keywords in English

human embryonic stem cells; CRISPR/Cas9; gene engineering; knockout

Tags

Tags

International impact, Reviewed
Změněno: 15/3/2018 17:02, Soňa Böhmová

Abstract

V originále

Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols

Links

GJ15-18316Y, research and development project
Name: Úloha microRNA v řízení buněčného dělení a diferenciace lidských embryonálních kmenových buněk do neurálních kmenových buněk. (Acronym: miRNA v diferenciaci lidských EK buněk)
Investor: Czech Science Foundation
GJ16-03269Y, research and development project
Name: Tau tubulin kináza 2 v ciliogenezi: molekulární mechanismy a funkční důsledky
Investor: Czech Science Foundation
MUNI/A/1369/2016, interní kód MU
Name: Zdroje pro tkáňové inženýrství 7 (Acronym: TissueENG 7)
Investor: Masaryk University, Category A
MUNI/C/1709/2016, interní kód MU
Name: Modeling Alzheimer´s disease in 3D human neural stem cells models
Investor: Masaryk University, Rector's Program
ROZV/24/LF/2016, interní kód MU
Name: LF - Příspěvek IP 2016
Investor: Ministry of Education, Youth and Sports of the CR
ROZV/25/LF/2017, interní kód MU
Name: LF - Příspěvek na IP 2017
Investor: Ministry of Education, Youth and Sports of the CR, Internal development projects