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@article{1395493,
author = {Bohačiaková, Dáša and Renzová, Tereza and Fedorová, Veronika and Barák, Martin and Bosáková, Michaela and Hampl, Aleš and Čajánek, Lukáš},
article_location = {New York},
article_number = {21},
doi = {http://dx.doi.org/10.1089/scd.2017.0058},
keywords = {human embryonic stem cells; CRISPR/Cas9; gene engineering; knockout},
language = {eng},
issn = {1547-3287},
journal = {Stem Cells and Development},
title = {An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System},
url = {http://online.liebertpub.com/doi/10.1089/scd.2017.0058},
volume = {26},
year = {2017}
}
TY - JOUR
ID - 1395493
AU - Bohačiaková, Dáša - Renzová, Tereza - Fedorová, Veronika - Barák, Martin - Bosáková, Michaela - Hampl, Aleš - Čajánek, Lukáš
PY - 2017
TI - An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System
JF - Stem Cells and Development
VL - 26
IS - 21
SP - 1521-1527
EP - 1521-1527
PB - Mary Ann Liebert, Inc.
SN - 15473287
KW - human embryonic stem cells
KW - CRISPR/Cas9
KW - gene engineering
KW - knockout
UR - http://online.liebertpub.com/doi/10.1089/scd.2017.0058
N2 - 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
ER -
BOHAČIAKOVÁ, Dáša, Tereza RENZOVÁ, Veronika FEDOROVÁ, Martin BARÁK, Michaela BOSÁKOVÁ, Aleš HAMPL and Lukáš ČAJÁNEK. An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System. \textit{Stem Cells and Development}. New York: Mary Ann Liebert, Inc., 2017, vol.~26, No~21, p.~1521-1527. ISSN~1547-3287. Available from: https://dx.doi.org/10.1089/scd.2017.0058.
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