Front. Cell Dev. Biol.

Sec. Stem Cell Research

Volume 12 - 2024 | doi: 10.3389/fcell.2024.1484955

This article is part of the Research Topic Pluripotent Stem Cells: Maintenance, Differentiation, and Applications View all 6 articles

Provisionally accepted

Paolo Petazzi 1 Francisco Gutierrez-Agüera 1* Heleia Roca-Ho 1* Julio Castaño 1 Clara Bueno 1 Niuska Alvarez 2* Lesley Forrester 3 Ana Sevilla 2* Antonella Fidanza 3* Pablo Menendez 1* 1 Josep Carreras Leukaemia Research Institute (IJC), Badalona, Catalonia, Spain 2 University of Barcelona, Barcelona, Spain 3 Centre for Regenerative Medicine (CRM), Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, Scotland, United Kingdom

The final, formatted version of the article will be published soon.

You have multiple emails registered with Frontiers:

Please enter your email address:

If you already have an account, please login

You don't have a Frontiers account ? You can register here

The CRISPR/Cas9 system has transformed genome editing by enabling precise modifications for diverse applications. Recent advancements, including base editing and prime editing, have expanded its utility beyond conventional gene knock-out and knock-in strategies. Additionally, several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains have been developed to modulate endogenous gene expression when directed to their regulatory regions by specific single-guide RNAs.Here, we report the development of the H9 human pluripotent stem cell (hPSC) line expressing an inducible dCas9-SAM activator (H9-iCas9.SAM), designed to activate transcription of endogenous genes. The H9-iCas9.SAM cells were generated through targeted integration of an inducible CRISPR/Cas9-based gene activator cassette into the AAVS1 "safe-harbour" locus. Molecular analyses confirmed precise and specific integration, ensuring minimal off-target effects. Functional characterization revealed that H9-iCas9.SAM cells retain pluripotency and display inducible endogenous gene activation upon doxycycline treatment. The versatility of H9-iCas9.SAM cells was demonstrated in directed in vitro differentiation assays, yielding neural stem cells (ectoderm), hematopoietic progenitor cells (mesoderm), and hepatocytes (endoderm). This underscores their potential in developmental biology studies and cell therapy applications. The engineered H9-iCas9.SAM line provides a robust platform for investigating gene function and advancing next-generation cell-based therapies.

Keywords: Human PSCs, ISAM, dead-Cas9 activation, Gene Expression, AAVS1 safe harbor locus

Received: 22 Aug 2024; Accepted: 11 Nov 2024.

Copyright: © 2024 Petazzi, Gutierrez-Agüera, Roca-Ho, Castaño, Bueno, Alvarez, Forrester, Sevilla, Fidanza and Menendez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Francisco Gutierrez-Agüera, Josep Carreras Leukaemia Research Institute (IJC), Badalona, 08916, Catalonia, Spain
Heleia Roca-Ho, Josep Carreras Leukaemia Research Institute (IJC), Badalona, 08916, Catalonia, Spain
Niuska Alvarez, University of Barcelona, Barcelona, Spain
Ana Sevilla, University of Barcelona, Barcelona, Spain
Antonella Fidanza, Centre for Regenerative Medicine (CRM), Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, EH16 4UU, Scotland, United Kingdom
Pablo Menendez, Josep Carreras Leukaemia Research Institute (IJC), Badalona, 08916, Catalonia, Spain

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.