2026-08-29T14:55:23Zhttps://repisalud.isciii.es/rest/oai/requestoai:repisalud.isciii.es:20.500.12105/92962024-11-29T17:12:20Zcom_20.500.12105_2173com_20.500.12105_2051col_20.500.12105_19597
Repisalud
author
Petazzi, Paolo
author
Torres-Ruiz Raul, Raul
author
Fidanza, Antonella
author
Roca-Ho, Heleia
author
Gutierrez-Agüera, Francisco
author
Castaño, Julio
author
Rodriguez Perales, Sandra
author
Díaz de la Guardia, Rafael
author
López-Millán, Belén
author
Bigas, Anna
author
Forrester, Lesley M
author
Bueno, Clara
author
Menéndez, Pablo
funder
Government of Catalonia (España)
funder
Fundación Josep Carreras Contra la Leucemia
funder
Fundación La Caixa
funder
Ministerio de Economía y Competitividad (España)
funder
Instituto de Salud Carlos III
funder
Asociación Española Contra el Cáncer
2020-03-20T18:08:48Z
2020-03-20T18:08:48Z
2020-02-27
Mol Ther Nucleic Acids. 2020;20:196-204.
10.1016/j.omtn.2020.02.009
2162-2531
Molecular therapy. Nucleic acids
32171171
http://hdl.handle.net/20.500.12105/9296
Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.
eng
CRISPR
SAM
SunTag
dCas9 activators
hMSCs
hPSCs
Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells
journal article
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