Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells
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 c...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-06-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253120300834 |
| _version_ | 1818191644441182208 |
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| author | Paolo Petazzi Raul Torres-Ruiz Antonella Fidanza Heleia Roca-Ho Francisco Gutierrez-Agüera Julio Castaño Sandra Rodriguez-Perales Rafael Díaz de la Guardia Belén López-Millán Anna Bigas Lesley M. Forrester Clara Bueno Pablo Menéndez |
| author_facet | Paolo Petazzi Raul Torres-Ruiz Antonella Fidanza Heleia Roca-Ho Francisco Gutierrez-Agüera Julio Castaño Sandra Rodriguez-Perales Rafael Díaz de la Guardia Belén López-Millán Anna Bigas Lesley M. Forrester Clara Bueno Pablo Menéndez |
| author_sort | Paolo Petazzi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T00:17:53Z |
| format | Article |
| id | doaj.art-f77cc3d8fc8949cb8341a5d7065e2798 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-12-12T00:17:53Z |
| publishDate | 2020-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-f77cc3d8fc8949cb8341a5d7065e27982022-12-22T00:44:48ZengElsevierMolecular Therapy: Nucleic Acids2162-25312020-06-0120196204Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem CellsPaolo Petazzi0Raul Torres-Ruiz1Antonella Fidanza2Heleia Roca-Ho3Francisco Gutierrez-Agüera4Julio Castaño5Sandra Rodriguez-Perales6Rafael Díaz de la Guardia7Belén López-Millán8Anna Bigas9Lesley M. Forrester10Clara Bueno11Pablo Menéndez12Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain; Corresponding author: Paolo Petazzi, PhD, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain.Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain; Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, SpainMRC Centre for Regenerative Medicine, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UKJosep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, SpainJosep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, SpainJosep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, SpainMolecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, SpainJosep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, SpainJosep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, SpainCancer Research Program, Institut Hospital del Mar d’Investigacions Mèdiques (IMIM), Barcelona, Spain; Centro de Investigación Biomédica en Red de Cáncer-CIBER-ONC, ISCIII, Barcelona, SpainMRC Centre for Regenerative Medicine, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UKJosep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red de Cáncer-CIBER-ONC, ISCIII, Barcelona, SpainJosep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red de Cáncer-CIBER-ONC, ISCIII, Barcelona, Spain; Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; Corresponding author: Pablo Menéndez, PhD, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain.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.http://www.sciencedirect.com/science/article/pii/S2162253120300834dCas9 activatorsSAMSunTaghPSCshMSCsCRISPR |
| spellingShingle | Paolo Petazzi Raul Torres-Ruiz Antonella Fidanza Heleia Roca-Ho Francisco Gutierrez-Agüera Julio Castaño Sandra Rodriguez-Perales Rafael Díaz de la Guardia Belén López-Millán Anna Bigas Lesley M. Forrester Clara Bueno Pablo Menéndez Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells Molecular Therapy: Nucleic Acids dCas9 activators SAM SunTag hPSCs hMSCs CRISPR |
| title | Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells |
| title_full | Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells |
| title_fullStr | Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells |
| title_full_unstemmed | Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells |
| title_short | Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells |
| title_sort | robustness of catalytically dead cas9 activators in human pluripotent and mesenchymal stem cells |
| topic | dCas9 activators SAM SunTag hPSCs hMSCs CRISPR |
| url | http://www.sciencedirect.com/science/article/pii/S2162253120300834 |
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