guest ::
| Home > Articles > Published articles > Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells |
| Home > Articles > Published articles > Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells |
(Institut Germans Trias i Pujol. Institut de Recerca contra la Leucèmia Josep Carreras) (Institut Germans Trias i Pujol. Institut de Recerca contra la Leucèmia Josep Carreras) (MRC Centre for Regenerative Medicine. University of Edinburgh) (Institut Germans Trias i Pujol. Institut de Recerca contra la Leucèmia Josep Carreras) (Institut Germans Trias i Pujol. Institut de Recerca contra la Leucèmia Josep Carreras) (Institut Germans Trias i Pujol. Institut de Recerca contra la Leucèmia Josep Carreras) (Centro Nacional de Investigaciones Oncológicas) (Institut Germans Trias i Pujol. Institut de Recerca contra la Leucèmia Josep Carreras) (Institut Hospital del Mar d'Investigacions Mèdiques) (Institut Germans Trias i Pujol. Institut de Recerca contra la Leucèmia Josep Carreras)| Date: | 2020 |
| Abstract: | 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. |
| Grants: | Ministerio de Economía y Competitividad SAF2016-80481-R Instituto de Salud Carlos III PI17-02303 Agència de Gestió d'Ajuts Universitaris i de Recerca SGR330 Agència de Gestió d'Ajuts Universitaris i de Recerca PERIS/2017-2019 Ministerio de Ciencia e Innovación BIO2017-91272-EXP Instituto de Salud Carlos III PI17-01028 |
| Note: | Altres ajuts: We thank Fundació Josep Carreras-Obra Social la Caixa for their institutional support. We thank Jose Luis Sardina (IJC, Barcelona) for technical assistance with the teratoma assays. Financial support for this work was obtained from the Fundación Leo Messi to P.M.; the Spanish Association against Cancer (AECC-CI-2015) ; and A.F. P.M. is an investigator of the Spanish Cell Therapy Cooperative Network (TERCEL). R.T.-R. is supported by a postdoctoral fellowship from the Asociación Española Contra el Cáncer (AECC). |
| Rights: | Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, i la comunicació pública de l'obra, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original. No es permet la creació d'obres derivades.  |
| Language: | Anglès |
| Document: | Article ; recerca ; Versió publicada |
| Subject: | CRISPR ; SAM ; SunTag ; DCas9 activators ; HMSCs ; HPSCs |
| Published in: | Molecular Therapy. Nucleic Acids, Vol. 20 (may 2020) , p. 196-204, ISSN 2162-2531 |
