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Developmental biology - Reprogramming Cells to Pluripotency|
A New way to turn skin cells into stem cells
"CRISPR/Cas9 can be used to activate genes... an attractive possibility for cellular reprogramming because multiple genes can be targeted at the same time. Reprogramming based on activation of endogenous genes rather than overexpression of transgenes is also, theoretically, a physiological way of controlling cell fate and may result in more normal cells. In this study, we show that it is possible to engineer a CRISPR activator system that allows robust reprogramming of iPSC."
"The technology may find practical use in bio banking and many other tissue technology applications, as it opens up new insights into the mechanisms controlling early embryonic gene activation," adds Jere Weltner, PhD student and first author of the article published in Nature Communications.
CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.
Authors: Jere Weltner, Diego Balboa, Shintaro Katayama, Maxim Bespalov, Kaarel Krjutškov, Eeva-Mari Jouhilahti, Ras Trokovic, Juha Kere and Timo Otonkoski.
We thank J. Saarimäki-Vire, S. Eurola, H. Grym, A. Laitinen, M. Salmela, Y. Novik, I. Fransson, and A. Damdimopoulos for technical assistance with the work and S. Vuoristo for comments on the manuscript. This work has been supported by the 3i Regeneration project (number 40395/13; a TEKES Large Strategic Research Opening), Jane and Aatos Erkko Foundation, Academy of Finland (No. 297466 and 312437, Center of Excellence in Stem Cell Metabolism), Sigrid Jusélius Foundation, Novo Nordisk Foundation, Instrumentarium Science Foundation, the Doctoral Program in Biomedicine at University of Helsinki, Knut and Alice Wallenberg Foundation (KAW2015.0096), SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under project b2014069, Bioinformatics and Expression Analysis core facility (BEA) and The Mutation Analysis Core Facility (MAF) at the Karolinska University Hospital.
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Colonies of CRISPRa reprogrammed "induced pluripotent stem cells (iPSC).
Image: Otonkoski Lab, University of Helsinki.