Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators
CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we descri
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Introduction Somatic cells can be reprogrammed to pluripotency by ectopic expression of a set of reprogramming factors [1]. Reprogramming factors are conventionally delivered into the reprogramming cells as transgenes. Other means that do not rely on transgenic reprogramming factors have also been reported. These include, for example, reprogramming by RNA interference and small molecular inhibitors [2–4]. CRISPR-mediated gene activation (CRISPRa) relies on sequence-specific targeting of a gene promoter for its activation by a deactivated Cas9 ribonucleoprotein (dCas9), fused with an activator domain [5–8]. Using this approach, any individual silenced endogenous gene can be activated specifically. CRISPRa may therefore be useful in pluripotent reprogramming by its activation of pluripotency-associated factors, which are silenced in the starting cell population. Pluripotent reprogramming with the CRISPRa system has been reported recently for both mouse and human [9, 10]. CRISPRa has the capacity to specifically activate the master pluripotency genes and subsequently the pluripotency regulatory networks.
Kejin Hu (ed.), Nuclear Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 2239, https://doi.org/10.1007/978-1-0716-1084-8_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Jere Weltner and Ras Trokovic
The most commonly used factors for human pluripotent reprogramming are OCT4 (POU5F1), SOX2, KLF4, MYC, NANOG, and/or LIN28A [11–13]. These factors can be delivered as non-integrating vectors to generate human iPSCs with their genomes intact. Commonly used non-integrating approaches for reprogramming include Sendai Viral replicons, replicating episomal plasmids, and modified mRNA transfections [14–16]. The method described herein utilizes the replicating but transient episomal plasmids to deliver a dCas9 activator and multiplexed guide RNA cassettes [10, 17]. Gene activation is achieved by using a dCas9 fused with a multimeric VP16 acidic activation domain (dCas9VP192) [18], and a plasmid with the U6 promoter to drive expression of guide RNAs targeting the promoters of OCT4, SOX2, KLF4, MYC, and LIN28A. An additional plasmid containing guide RNA sequences targeting a conserved EGA-enriched Alu-motif (EEA-motif) is included to enhance the reprogramming efficiency [10, 19]. The core protocol for reprogramming human fibroblasts with CRISPRa is described in the first part of the methods section (Subheadings 3.1–3.7). Additionally, we have included methods for assembling and cloning of multiplexed guide RNA plasmids for readers to prepare their own targeting constructs (Subheadings 3.8 and 3.9). The second part has been included in the case that the core protocol needs to be modified, for example, to generate new guides targeting additional genes.
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Materials
2.1 Cells and Episomal Vectors for Reprogramming
1. Human foreskin fibroblasts (HFF). 2. Activator plasmid: pCXLE-dCas9VP192-T2A-EGFP-shP53, Addgene plasmid #, 69535 (see Note 1). 3. Guide RNA plasmid tar
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