RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts Kai Xiong, 1, * Yan Zhou, 2, * Kristian Aabo Blichfeld, 2, * Poul Hyttel, 1 Lars Bolund, 2 Kristine Karla Freude, 1 and Yonglun Luo 2 Abstract Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRIS- PRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts). Activation potential was monitored using a luciferase reporter system and we found that both CRISPRa and SAM can efficiently activate the proximal promoter of all five genes. We also observed that the guide RNA (gRNA) target sites and number of gRNAs have a major effect on gRNA-guided activation efficiency. Furthermore, increased activation efficiency (>3-folds) could be achieved by the SAM system compared to CRISPRa. In addition, we discovered that only the SAM system could efficiently activate LIN28, OCT4, and SOX2 expression (up to 100-folds compared to coexpression with a scrambled gRNA) in primary human fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular reprogramming using ATFs. Keywords: CRISPR, pluripotency genes, induced pluripotent stem cells, activation, reprogramming Introduction E ctopic expression of the four pluripotency fac- tors, OCT4, SOX2, MYC, and KLF4, is sufficient to reprogram mature cells from any given cell type into human induced pluripotent stem cells (iPSC) (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007). However, this strategy is faced with various challenges. Exogenous genes might integrate into the genome of the target cells, which could hamper further utilization of iPSCs (Sel- varaj et al., 2010). Moreover, if endogenous pluripotent genes are not fully activated by the exogenous factors, an incom- plete reprogramming can occur, which has consequences for their downstream usage and differentiation potential. An alternative approach would be the direct activation of the endogenous pluripotency genes instead of delivering the reprogramming factors exogenously. Previous studies have reported that human endogenous pluripotency genes, in- cluding OCT4, SOX2, MYC, and KLF4, can be activated by artificial transcription factors (ATFs) via zinc fingers ( Ji et al., 2014) or transcription activator-like effectors (TALEs) tech- nology (Chavez et al., 2015; Gao et al., 2013; Hu et al., 2014). However, the broader application of these technologies is limited by the complexity and uncertainty of programmable vector generation and unpredicted targeting efficiency. Recently, the clustered regularly interspaced short palin- dromic repeats (CRISPR) and nuclease-deficient CRISPR- associated protein 9 (dCas9)-based systems have been applied for ATFs construction and regulation of gene ex- pression (Gilbert et al., 2013; Qi et al., 2013). By fusing dCas9 to a transcription effector factor domain such as VP64, the dCas9-VP64 ATF can specifically bind to any 1 Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark. 2 Danish Regenerative Engineering Alliance for Medicine, Department of Biomedicine, Aarhus University, Aarhus, Denmark. *These authors contributed equally to this work. CELLULAR REPROGRAMMING Volume 19, Number 3, 2017 ª Mary Ann Liebert, Inc. DOI: 10.1089/cell.2017.0006 189