2026-05-22T00:56:09Zhttps://repisalud.isciii.es/rest/oai/request

oai:repisalud.isciii.es:20.500.12105/92962024-11-29T17:12:20Zcom_20.500.12105_2173com_20.500.12105_2051col_20.500.12105_19597

00925njm 22002777a 4500 dc 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 author 2020-02-27 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. 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 CRISPR SAM SunTag dCas9 activators hMSCs hPSCs Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells