An efficient method for generation of Knockout human embryonic stem cells using CRISPR/Cas9 system

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F17%3A00067189" target="_blank" >RIV/00159816:_____/17:00067189 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14110/17:00095168

Výsledek na webu

<a href="http://dx.doi.org/10.1089/scd.2017.0058" target="_blank" >http://dx.doi.org/10.1089/scd.2017.0058</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1089/scd.2017.0058" target="_blank" >10.1089/scd.2017.0058</a>

Jazyk výsledku

angličtina

Název v původním jazyce

An efficient method for generation of Knockout human embryonic stem cells using CRISPR/Cas9 system

Popis výsledku v původním jazyce

Human embryonic stem cells (hESCs) represent 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/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 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 hESC 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.

Název v anglickém jazyce

An efficient method for generation of Knockout human embryonic stem cells using CRISPR/Cas9 system

Popis výsledku anglicky

Human embryonic stem cells (hESCs) represent 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/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 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 hESC 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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

30205 - Hematology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2017

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Stem Cells and Development

ISSN

1547-3287

e-ISSN

—

Svazek periodika

26

Číslo periodika v rámci svazku

21

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

7

Strana od-do

1521-1527

Kód UT WoS článku

000413639900001

EID výsledku v databázi Scopus

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