Robustness of catalytically dead Cas9 activators in human pluripotent and mesenchymal stem cells

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  • dc.contributor.author Petazzi, Paolo
  • dc.contributor.author Torres-Ruiz, Raul
  • dc.contributor.author Fidanza, Antonella
  • dc.contributor.author Roca-Ho, Heleia
  • dc.contributor.author Gutierrez-Agüera, Francisco
  • dc.contributor.author Castaño, Julio
  • dc.contributor.author Rodríguez-Perales, Sandra
  • dc.contributor.author Diaz de la Guardia, Rafael
  • dc.contributor.author López-Millán, Belén
  • dc.contributor.author Bigas Salvans, Anna
  • dc.contributor.author Forrester, Lesley M.
  • dc.contributor.author Bueno, Clara
  • dc.contributor.author Menéndez, Pablo
  • dc.date.accessioned 2020-06-11T07:14:52Z
  • dc.date.available 2020-06-11T07:14:52Z
  • dc.date.issued 2020
  • dc.description.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.
  • dc.description.sponsorship We thank CERCA/Generalitat de Catalunya and 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 Catalunya Goverment (SGR330 and PERIS 2017-2019), the Spanish Ministry of Economy and Competitiveness (SAF2016-80481-R), the European Research Council (CoG-2014-646903), and the Fundación Leo Messi to P.M.; the Spanish Association against Cancer (AECC-CI-2015) and the Health Institute Carlos III (ISCIII/FEDER, PI17/01028) to C.B.; the Biotechnology and Biological Sciences Research Council (BBRSC) to L.M.F. and A.F.; and the Spanish National Research and Development Plan (ISCIII/FEDER, PI17/02303) and the AEI/MICIU EXPLORA Project (BIO2017-91272-EXP) to S.R.-P. 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).
  • dc.format.mimetype application/pdf
  • dc.identifier.citation Petazzi P, Torres-Ruiz R, Fidanza A, Roca-Ho H, Gutierrez-Agüera F, Castaño J, et al. Robustness of catalytically dead Cas9 activators in human pluripotent and mesenchymal stem cells. Mol Ther Nucleic Acids. 2020 Jun 5; 20:196-204. DOI: 10.1016/j.omtn.2020.02.009
  • dc.identifier.doi http://dx.doi.org/10.1016/j.omtn.2020.02.009
  • dc.identifier.issn 2162-2531
  • dc.identifier.uri http://hdl.handle.net/10230/44954
  • dc.language.iso eng
  • dc.publisher Elsevier
  • dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/ 646903
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/SAF2016-80481-R
  • dc.rights Copyright © 2020 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
  • dc.rights.accessRights info:eu-repo/semantics/openAccess
  • dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/4.0/
  • dc.subject.keyword CRISPR
  • dc.subject.keyword SAM
  • dc.subject.keyword SunTag
  • dc.subject.keyword dCas9 activators
  • dc.subject.keyword hMSCs
  • dc.subject.keyword hPSCs
  • dc.title Robustness of catalytically dead Cas9 activators in human pluripotent and mesenchymal stem cells
  • dc.type info:eu-repo/semantics/article
  • dc.type.version info:eu-repo/semantics/publishedVersion

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