We also demonstrate stable epigenetic remodeling of the locus and uncover transcriptome-wide differences that result from endogenous versus exogenous expression

We also demonstrate stable epigenetic remodeling of the locus and uncover transcriptome-wide differences that result from endogenous versus exogenous expression. controlling cell-fate decisions. without relinquishing their stemness, resulting in loss of engraftment capabilities (Montarras, 2005). Consequently, the generation of functional PAX7+ satellite cells from hPSCs has been attempted by pairing various differentiation protocols with exogenous PAX7 cDNA overexpression (Darabi et?al., 2012, Kim et?al., 2017, Rao et?al., 2018). Here, we explore an alternative strategy of generating myogenic progenitor cells via activation of the endogenous gene. Advances in genome-engineering technologies have established the type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system as a programmable transcriptional regulator capable of targeted activation or repression of endogenous genes. Mutations to the catalytic residues of the Cas9 protein results in a nuclease-deactivated Cas9 (dCas9) that can be fused to various effector domains to exert their function on precise genomic loci defined by the guide RNA (gRNA) (Gilbert et?al., 2013, Jinek et?al., 2012). For example, fusion of dCas9 to the transactivation domain VP64 can potently activate genes in their native chromosomal context when gRNAs are designed at target gene promoters (Maeder et?al., 2013, Perez-Pinera et?al., 2013). In contrast to ectopic expression Rabbit Polyclonal to VIPR1 of transgenes, activation of endogenous genes facilitates chromatin remodeling and Sulindac (Clinoril) induction of autonomously maintained gene networks (Black et?al., 2016, Chakraborty et?al., 2014, Liu et?al., 2018a). Targeting endogenous genes can also capture the full complexity of transcript isoforms, mRNA localization, and other effects of non-coding regulatory elements, which may be critical for proper cellular reprogramming. We and several other groups have demonstrated cellular reprogramming with CRISPR/Cas9-based transcriptional regulators in the context of somatic cell reprogramming (Balboa et?al., 2015, Black et?al., 2016, Chakraborty et?al., 2014, Liu et?al., 2018a, Weltner et?al., 2018) as well as directed differentiation of pluripotent stem cells (PSCs) into various cell types (Balboa et?al., 2015, Chavez et?al., 2015, Kearns et?al., 2014, Liu et?al., 2018b). However, there has not yet been a demonstration of differentiation of hPSCs with CRISPR/Cas9-based transcriptional activators to generate cells capable of transplantation, engraftment, and tissue regeneration. In this study, we use VP64dCas9VP64 to activate the Sulindac (Clinoril) endogenous gene encoding the transcription factor PAX7 in both human embryonic stem cells (ESCs) and induced PSCs (iPSCs) to direct differentiation into skeletal muscle progenitors. We demonstrate the derivation of functional skeletal muscle progenitor cells that can be induced to differentiate and can also participate in regeneration of damaged muscles when transplanted into mice. We also demonstrate stable epigenetic remodeling of the locus and uncover transcriptome-wide differences that result from endogenous versus Sulindac (Clinoril) exogenous expression. These results establish CRISPR-based activation of gene networks governing progenitor cell specification as a potential strategy for cell therapy and regenerative medicine. Results Developing Conditions for VP64dCas9VP64-Mediated Endogenous Activation in hPSCs During embryonic differentiation, PAX7 and its paralog PAX3 specify myogenic cells within the paraxial mesoderm. Differentiation of hPSCs into paraxial mesoderm cells can be initiated by CHIR99021, a GSK3 inhibitor (Tan et?al., 2013). Two human pluripotent stem cell lines, H9 ESCs and DU11 iPSCs, were used for differentiation studies. For targeted gene activation, we used the dCas9 with the VP64 domain fused to both the N and C termini (VP64dCas9VP64), which we previously showed to be 10-fold more potent than a solitary VP64 fusion (Black et?al., 2016, Chakraborty et?al., 2014). To test the effectiveness of VP64dCas9VP64-mediated activation of gene (Number?S1A). H9 Sulindac (Clinoril) ESCs stably expressing VP64dCas9VP64 were differentiated into paraxial mesoderm cells with addition of CHIR99021 in E6 medium for 2?days, while previously described (Shelton et?al., 2014). Cells were transfected with the individual gRNAs and samples were harvested 6?days later on for gene-expression analysis using qRT-PCR. Four out of the eight gRNAs significantly upregulated compared with mock transfected cells (Number?S1B). In a second screen, we packaged the four individual gRNAs that performed best in the transfection experiment into lentiviruses to accomplish more stable and Sulindac (Clinoril) robust manifestation. Cells were harvested at 8?days post transduction. gRNA #4 was identified as the most potent gRNA.