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Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells

Paolo Petazzi1, Raul Torres-Ruiz2, Antonella Fidanza3

  • 1Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.

Molecular Therapy. Nucleic Acids
|March 15, 2020
PubMed

Insights

Second-generation CRISPR activators, dCas9-SAM and dCas9-SunTag, effectively activate multiple genes simultaneously in human stem cells. These advanced systems offer robust and specific gene expression for cellular therapies and disease modeling.

Area of Science:

  • * Molecular Biology
  • * Gene Regulation
  • * Stem Cell Biology

Background:

  • * Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are crucial for cellular therapies and disease modeling.
  • * Modifying cell fate requires simultaneous activation of multiple endogenous genes, which is challenging in these cells.
  • * Catalytically dead Cas9 (dCas9) fused to activation domains, guided by sgRNAs, can modulate gene expression.

Purpose of the Study:

  • * To compare the efficacy of first-generation (dCas9-VP64) and second-generation (dCas9-SAM, dCas9-SunTag) dCas9 activators for gene induction in hPSCs and hMSCs.
  • * To evaluate single and multiplexed gene activation capabilities of these systems.
  • * To assess the robustness and specificity of dCas9 activators in clinically relevant human stem cells.

Main Methods:

  • * Testing of dCas9-VP64, dCas9-SAM, and dCas9-SunTag systems for gene activation in various hPSC and hMSC lines.
  • * Application of single-guide RNA (sgRNA) to direct dCas9 activators to target gene regulatory regions.
  • * Comparison of gene expression levels following single and multiplexed gene activation strategies.

Main Results:

  • * All three dCas9 activator systems induced specific and potent gene expression in both single and multiplexed settings.
  • * Second-generation systems (dCas9-SAM and dCas9-SunTag) demonstrated superior and more consistent gene expression levels compared to dCas9-VP64.
  • * Simultaneous targeting of the same gene with multiple sgRNAs did not yield additive gene expression increases in either hPSCs or hMSCs.

Conclusions:

  • * Second-generation dCas9 activators (dCas9-SAM and dCas9-SunTag) are highly effective tools for simultaneous gene activation in human stem cells.
  • * These systems offer robust and specific gene expression modulation, crucial for advancing cellular therapies and research.
  • * The study validates the utility of dCas9-SAM and dCas9-SunTag for genetic manipulation in clinically relevant stem cell applications.
Keywords:
CRISPRSAMSunTagdCas9 activatorshMSCshPSCs

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