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• W2783807303 abstract "•Endogenous Oct4 and Sox2 can be targeted and activated by CRISPR activation•Activation of endogenous Oct4 or Sox2 triggers reprogramming to pluripotency•Oct4 promoter and enhancer are simultaneously remodeled by dCas9-SunTag-p300core•Authentic induced pluripotent stem cells are generated with CRISPR activation Generation of induced pluripotent stem cells typically requires the ectopic expression of transcription factors to reactivate the pluripotency network. However, it remains largely unclear what remodeling events on endogenous chromatin trigger reprogramming toward induced pluripotent stem cells (iPSCs). Toward this end, we employed CRISPR activation to precisely target and remodel endogenous gene loci of Oct4 and Sox2. Interestingly, we found that single-locus targeting of Sox2 was sufficient to remodel and activate Sox2, which was followed by the induction of other pluripotent genes and establishment of the pluripotency network. Simultaneous remodeling of the Oct4 promoter and enhancer also triggered reprogramming. Authentic pluripotent cell lines were established in both cases. Finally, we showed that targeted manipulation of histone acetylation at the Oct4 gene locus could also initiate reprogramming. Our study generated authentic iPSCs with CRISPR activation through precise epigenetic remodeling of endogenous loci and shed light on how targeted chromatin remodeling triggers pluripotency induction. Generation of induced pluripotent stem cells typically requires the ectopic expression of transcription factors to reactivate the pluripotency network. However, it remains largely unclear what remodeling events on endogenous chromatin trigger reprogramming toward induced pluripotent stem cells (iPSCs). Toward this end, we employed CRISPR activation to precisely target and remodel endogenous gene loci of Oct4 and Sox2. Interestingly, we found that single-locus targeting of Sox2 was sufficient to remodel and activate Sox2, which was followed by the induction of other pluripotent genes and establishment of the pluripotency network. Simultaneous remodeling of the Oct4 promoter and enhancer also triggered reprogramming. Authentic pluripotent cell lines were established in both cases. Finally, we showed that targeted manipulation of histone acetylation at the Oct4 gene locus could also initiate reprogramming. Our study generated authentic iPSCs with CRISPR activation through precise epigenetic remodeling of endogenous loci and shed light on how targeted chromatin remodeling triggers pluripotency induction. Pluripotent stem cells hold great promise for regenerative medicine. A better understanding of how endogenous chromatin remodeling leads to pluripotency induction is of significant interest. Conventionally, differentiated somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18864) Google Scholar). Overexpressed Oct4, Sox2, and Klf4 initially bind to and globally remodel endogenous loci across the genome (Soufi et al., 2012Soufi A. Donahue G. Zaret K.S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome.Cell. 2012; 151: 994-1004Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar), ultimately leading to establishment of pluripotent regulatory circuitry. However, it is largely unknown what precise remodeling events on endogenous chromatin trigger reprogramming toward pluripotency. First of all, whether simultaneous remodeling of a large number of pluripotency-related loci is necessary or precise remodeling of a single locus is sufficient for iPSC induction is not clear. Besides, Oct4, Sox2, and Klf4 target the distal elements of many genes required for reprogramming (Soufi et al., 2012Soufi A. Donahue G. Zaret K.S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome.Cell. 2012; 151: 994-1004Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar), but how the remodeling of these distal elements would affect pluripotency induction is poorly understood. Furthermore, epigenetic remodeling is the central mechanism of cellular reprogramming (Smith et al., 2016Smith Z.D. Sindhu C. Meissner A. Molecular features of cellular reprogramming and development.Nat. Rev. Mol. Cell Biol. 2016; 17: 139-154Crossref PubMed Scopus (108) Google Scholar), but it has not been determined whether iPSC induction can be initiated by epigenetic manipulation of any defined endogenous loci. Single-cell analysis and computational modeling suggested that activation of endogenous Sox2 gene marked a deterministic event to pluripotency, likely triggering reprogramming toward iPSCs (Buganim et al., 2012Buganim Y. Faddah D.A. Cheng A.W. Itskovich E. Markoulaki S. Ganz K. Klemm S.L. van Oudenaarden A. Jaenisch R. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase.Cell. 2012; 150: 1209-1222Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). However, due to the methodological limitations, there is no direct evidence of whether pluripotency can be induced by precise remodeling of Sox2 locus. Recently, the type II clustered regularly interspaced short palindromic repeat and Cas9 nuclease (CRISPR/Cas9) system from bacteria was repurposed as a powerful tool for genome editing in mammalian cells (Cong et al., 2013Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9981) Google Scholar, Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9327) Google Scholar, Mali et al., 2013bMali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6424) Google Scholar). A deactivated form of Cas9, dead Cas9 (dCas9), has been engineered as programmable synthetic transcription factors when fused with transactivation domains, which is termed the CRISPR activation (CRISPRa) system (Chavez et al., 2015Chavez A. Scheiman J. Vora S. Pruitt B.W. Tuttle M. P R Iyer E. Lin S. Kiani S. Guzman C.D. Wiegand D.J. et al.Highly efficient Cas9-mediated transcriptional programming.Nat. Methods. 2015; 12: 326-328Crossref PubMed Scopus (887) Google Scholar, Gilbert et al., 2013Gilbert L.A. Larson M.H. Morsut L. Liu Z. Brar G.A. Torres S.E. Stern-Ginossar N. Brandman O. Whitehead E.H. Doudna J.A. et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.Cell. 2013; 154: 442-451Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar, Konermann et al., 2015Konermann S. Brigham M.D. Trevino A.E. Joung J. Abudayyeh O.O. Barcena C. Hsu P.D. Habib N. Gootenberg J.S. Nishimasu H. et al.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.Nature. 2015; 517: 583-588Crossref PubMed Scopus (1661) Google Scholar, Tanenbaum et al., 2014Tanenbaum M.E. Gilbert L.A. Qi L.S. Weissman J.S. Vale R.D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging.Cell. 2014; 159: 635-646Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar, Zalatan et al., 2015Zalatan J.G. Lee M.E. Almeida R. Gilbert L.A. Whitehead E.H. La Russa M. Tsai J.C. Weissman J.S. Dueber J.E. Qi L.S. Lim W.A. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds.Cell. 2015; 160: 339-350Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar). This system reportedly can function as a pioneer factor to target the silenced chromatin locus with high precision and promote downstream gene transcription (Polstein et al., 2015Polstein L.R. Perez-Pinera P. Kocak D.D. Vockley C.M. Bledsoe P. Song L. Safi A. Crawford G.E. Reddy T.E. Gersbach C.A. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.Genome Res. 2015; 25: 1158-1169Crossref PubMed Scopus (94) Google Scholar). Moreover, Hilton and colleagues showed that dCas9-p300core fusion protein can be used to manipulate the histone acetylation of targeted genomic sites (Hilton et al., 2015Hilton I.B. D’Ippolito A.M. Vockley C.M. Thakore P.I. Crawford G.E. Reddy T.E. Gersbach C.A. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.Nat. Biotechnol. 2015; 33: 510-517Crossref PubMed Scopus (1156) Google Scholar). With these features, the CRISPRa system provides an advantageous tool to precisely remodel endogenous chromatin loci for cellular reprogramming (Black et al., 2016Black J.B. Adler A.F. Wang H.G. D’Ippolito A.M. Hutchinson H.A. Reddy T.E. Pitt G.S. Leong K.W. Gersbach C.A. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells.Cell Stem Cell. 2016; 19: 406-414Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, Chakraborty et al., 2014Chakraborty S. Ji H. Kabadi A.M. Gersbach C.A. Christoforou N. Leong K.W. A CRISPR/Cas9-based system for reprogramming cell lineage specification.Stem Cell Reports. 2014; 3: 940-947Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In this study, using CRISPR activation, the SunTag system, we demonstrated that precise remodeling of endogenous Oct4 or Sox2 gene locus was sufficient to induce pluripotency. To determine whether and how remodeling of endogenous loci initiate reprogramming toward pluripotency, we used the SunTag system to precisely remodel endogenous pluripotency gene loci in mouse embryonic fibroblasts (MEFs). dCas9-SunTag-VP64 was chosen for its enhanced chromatin-remodeling activity by recruiting multiple VP64 to one targeting site (Figure 1A) (Tanenbaum et al., 2014Tanenbaum M.E. Gilbert L.A. Qi L.S. Weissman J.S. Vale R.D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging.Cell. 2014; 159: 635-646Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). dCas9 expression was controlled by a Tet-On promoter. Oct4 and Sox2 loci were selected as targets because of their central roles in pluripotency induction and maintenance. Single guide RNAs (sgRNAs) were designed to target the Oct4 and Sox2 promoters, as well as the Oct4 enhancer. Besides the activation effect of sgRNAs, multiple factors were considered, regarding the genomic sequences targeted, including their proximity to but no overlapping with the binding sites of pluripotent factor and transcription machinery, histone H3K27 acetylation in pluripotent stem cells, and their potential to form promoter-enhancer loops mediated by Mediator complex (Figures S1A–S1C). We first examined transcriptional activation of target genes with each designed Oct4 and Sox2 sgRNA delivered by lentivirus in differentiating mouse embryonic stem cells (ESCs) (Figure S1D). Mouse ESCs were first transduced with dCas9-SunTag-VP64 system and sgRNAs. Because Oct4 and Sox2 are highly expressed in ESCs, we induced ESC differentiation with 1 μM retinoic acid (RA). Meanwhile, the dCas9-SunTag-VP64 system was induced with doxycycline. Analysis of Oct4 expression showed that sgRNAs targeting a narrow promoter region close to the transcription start site (TSS), and a 200-bp region of distal enhancer can enhance the transcription (Figure S1E). As for the Sox2 promoter, sgRNA activity showed a remarkable tendency for higher gene activation with sgRNAs closer to the TSS (Figure S1E). Selected sgRNAs and the dCas9-SunTag-VP64 were also transduced into MEFs (Figure S1F). sgRNAs O-127 and O-71 targeting 127- and 71-bp upstream of Oct4 TSS were combined to target the promoter. Similarly, O-1965, O-2066, and O-2135 were combined to target the Oct4 enhancer; and separately, S-84, S-136, and S-148 were combined for Sox2 promoter targeting. After 4 days of dCas9 induction by doxycycline, targeting the Oct4 promoter led to about a 100-fold increase in Oct4 transcription, and targeting the enhancer resulted in modest activation (Figure S1G). For Sox2 promoter, about 15-fold activation was detected (Figure S1G). This suggests that, guided by specific sgRNAs, dCas9-SunTag-VP64 can activate the silenced Oct4 and Sox2 in MEFs. We next sought to determine whether pluripotency network can be fully reactivated and established in MEFs. We optimized the SunTag reprogramming system in two ways. First, more gene promoters were targeted by adding the corresponding sgRNAs. Klf4, c-Myc (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18864) Google Scholar), Nr5a2 (Heng et al., 2010Heng J.C. Feng B. Han J. Jiang J. Kraus P. Ng J.H. Orlov Y.L. Huss M. Yang L. Lufkin T. et al.The nuclear receptor Nr5a2 can replace Oct4 in the reprogramming of murine somatic cells to pluripotent cells.Cell Stem Cell. 2010; 6: 167-174Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), Glis1 (Maekawa et al., 2011Maekawa M. Yamaguchi K. Nakamura T. Shibukawa R. Kodanaka I. Ichisaka T. Kawamura Y. Mochizuki H. Goshima N. Yamanaka S. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1.Nature. 2011; 474: 225-229Crossref PubMed Scopus (304) Google Scholar), and Cebpa (Di Stefano et al., 2014Di Stefano B. Sardina J.L. van Oevelen C. Collombet S. Kallin E.M. Vicent G.P. Lu J. Thieffry D. Beato M. Graf T. C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells.Nature. 2014; 506: 235-239Crossref PubMed Scopus (153) Google Scholar) were selected. For each promoter, 4–10 sgRNAs were designed and tested in differentiating ESCs (Figure S1E). 1–3 sgRNAs for each promoter were included in the previous Oct4/Sox2 sgRNA pool (Table S1). Second, a small-molecule cocktail, consisting of Parnate, Chir99021, A83-01, and Forskolin (PCAF), was added into our reprogramming medium. This chemical cocktail further increased Oct4 and Sox2 transcription by 3–4 times on day 4 (Figure S1H). To monitor the reactivation of pluripotency network, we used OG2 MEF cells that harbor a stable Oct4-EGFP reporter and exhibit intense EGFP signal when endogenous Oct4 is actively transcribed (Szabó et al., 2002Szabó P.E. Hübner K. Schöler H. Mann J.R. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells.Mech. Dev. 2002; 115: 157-160Crossref PubMed Scopus (268) Google Scholar). After transduction of dCas9-SunTag-VP64 and the sgRNA pool (18 sgRNAs in total, Table S1), the MEF medium was changed to reprogramming medium with doxycycline. This was denoted as day 0 (Figure 1B). Since day 4, transcription of Oct4 and Sox2 became more and more robust (Figure 1D). By day 7, reprogramming clusters appeared, and after 2 weeks, EGFP-positive colonies were visible (Figure 1C). Those colonies were also positive for Nanog, Sox2, and SSEA-1 (Figure 1F). Then, EGFP-positive colonies were expanded on feeder cells to generate CRISPR iPSC lines. Those CRISPR iPSCs formed typical mouse ES-like domed colonies with a strong EGFP signal (Figure 1E). A panel of pluripotency genes, including Oct4, Sox2, Nanog, Esrrb, Nr5a2, and Utf1, was highly expressed (Figure 1G). These cells can be passaged for more than 20 passages without any sign of losing the EGFP signal or ES morphology (Figure 1E). These data demonstrate that pluripotency has been established in these CRISPR iPSCs. To identify the essential loci required for CRISPR iPSC generation, we removed sgRNAs targeting each individual locus one by one from the pool. In this 18-sgRNA pool, removal of sgRNAs targeting the Oct4, Sox2, or Glis1 promoter or the Oct4 enhancer led to a sharp decrease in the number of EGFP-positive colonies (Figure S2A), indicating potential roles for these loci in pluripotency induction. Next, we determined whether targeting of Oct4, Sox2, and Glis1 promoters together was sufficient to generate iPSCs. Since single sgRNAs could achieve gene activation at the level of 60% to even 180% of their corresponding two- or three-sgRNA combinations (Figure S2B), we selected one sgRNA to target each promoter, O-127 for Oct4, S-84 for Sox2, and G-215 for Glis1. This simplified our system and potentially decreased off-target effect. The combination of OSG (O-127, S-84, and G-215) could activate the three genes properly (Figure S2C). After 2 weeks, EGFP-positive colonies were observed, and iPSC lines could be established (Figures S2D and S2E). During OSG reprogramming, we surprisingly noticed that EGFP-positive colonies appeared when S-84 alone was used (Figure S2F), suggesting that targeting Sox2 promoter alone may be sufficient for pluripotency induction. To rule out the possibility of an off-target effect from S-84, we examined the top 10 predicted targets of S-84, and only the Sox2 gene was significantly activated (Figure S2G). Sox2 protein was also detected on day 4 (Figure 2B). Besides, we repeated the reprogramming tests with another two Sox2 sgRNAs, S-136 and S-148 (Figure 2A). These two sgRNAs individually activated endogenous Sox2 transcription (Figure 2C), and EGFP-positive colonies were obtained (Figure 2D). Then, we examined whether the iPSCs were authentic pluripotent. Within these EGFP-positive colonies, Nanog and SSEA-1 protein was also detected, and CRISPR iPSC lines were established (Figures 2E and 2F). For line S-17, expression of key pluripotent factors was similar to that in R1 cells (Figure 2G). These cells were also karyotypically normal (Figure 2H). A more stringent assay for pluripotency was performed. S-17 cells were injected into the blastocysts of B6(Cg)-Tyrc-2J/J (B6-albino) background, and EGFP-positive cells were found in the gonadal regions of 71.4% (5 out of 7) E13.5 embryos (Figure 2I). Live-born chimeras were generated (Figure 2J), and the rate was 46.2% (6 out of 13). More importantly, the S-17 cells were also germline competent (Figure 2K). With these data, we concluded that single-locus targeting of the Sox2 promoter by one sgRNA was sufficient to reprogram MEFs into authentic pluripotent stem cells. With the lentiviral transduction, the reprogramming efficiency was relatively low and variable in both OG2 and 129 background MEFs (0%–0.013%) (Figures 2C, S2A, S2F, and S3A–C). This may be from inefficient delivery of SunTag components and random copy numbers of the components delivered in single cells. This was reflected by the varied copy numbers of sgRNA cassette in the genomes of established iPSC lines, and one to five copies were found per cell among 12 lines (Figure S3D). To decrease the variability and enhance the efficiency, we decided to generate secondary MEFs using a CRISPR iPSC line that was derived from single colony. S-17 iPSCs were labeled with blue fluorescence protein (BFP) and injected into B6 blastocysts, and secondary MEFs were derived from the E13.5 embryos (Figure 3A). About half of the MEFs (52.4%) were originated from the S-17 iPSCs revealed by flow cytometry (Figure S3E), and these secondary MEF cells were termed S-17 MEFs. With doxycycline, endogenous Sox2 was readily detected by both qPCR and immune-fluorescent staining, but no Sox2 was detected without doxycycline (Figures 3B and 3D). No off-target genes were dramatically elevated (Figure S3G). These data demonstrate that the SunTag system functioned properly to activate Sox2 in S-17 MEFs. Then the S-17 MEFs were examined if they were reprogrammable. Sox2 transcription was significantly upregulated on day 4 and increased quickly to R1 mouse ES level by day 8 (Figure 3B). Following Sox2 upregulation, other core pluripotent factors, Oct4, Nanog, and Rex1, were also activated. Their transcription was detected on day 8 and elevated dramatically after that (Figure 3G). Meanwhile, morphological changes were observed from day 4, and EGFP-positive colonies were visible on day 7 (Figure 3E). iPSC lines could also be established (Figure 3E). With S-17 MEFs, the reprogramming efficiency (0.1%) increased by 40-fold over the lentivirus method (Figure 3F). As expected, much less variability was observed (Figure S3H). We also tested whether more differentiated tail tip fibroblasts (TTFs) were reprogrammable. We derived S-17 TTFs from the 14-month-old chimeric mouse. In presence of doxycycline, TTFs underwent morphological changes, and EGFP-positive colonies were obtained in 2 weeks (Figure S3L). These observations show that S-17 MEFs and TTFs were reprogrammable. Without doxycycline, we could not see the activation of Sox2, and no colonies were obtained (Figures 3B and 3J). When the PCAF cocktail was removed, EGFP-positive colonies were still generated (Figure S3I), although with lower efficiency. Based on this, we concluded that endogenous Sox2 activation was the trigger for S-17 MEF reprogramming. Then, we examined whether the reprogramming was dose dependent on Sox2 level. Sox2 was activated with a series of doxycycline concentrations (e.g., 0, 0.01, 0.1, and 1 μg/mL). We noticed that Sox2 level showed a positive correlation with the dox concentrations, and the reprogramming efficiency was clearly dependent on Sox2 level (Figure 3J). VP64 promotes gene transcription and chromatin remodeling by recruiting multiple epigenetic modifiers (Hirai et al., 2010Hirai H. Tani T. Kikyo N. Structure and functions of powerful transactivators: VP16, MyoD and FoxA.Int. J. Dev. Biol. 2010; 54: 1589-1596Crossref PubMed Scopus (61) Google Scholar), so we tested how the SunTag system epigenetically remodeled the Sox2 promoter. Chromatin immunoprecipitation (ChIP) was performed with H3K27 acetylation (H3K27ac) antibody against the Sox2 promoter. As early as day 4, the H3K27ac level was already elevated 2-fold, and it further increased on days 8 and 12 (Figure 3C). This indicates that the SunTag targeting caused gradual and constant epigenetic remodeling at the Sox2 promoter. We also checked the promoters of Oct4, Nanog, and Rex1, and their H3K27ac levels increased significantly with a 4-day latency, similar to the gene transcription (Figures 3G and 3H). Interestingly, the enhancers of Oct4 showed simultaneous elevation of H3K27ac level (Figure 3I). These data suggest that activation of Sox2 facilitated following induction of other key genes for pluripotency establishment. We then tested whether additional targeting of the Oct4 promoter in S-17 MEFs would promote the reprogramming efficiency. Transduction of O-127 led to a significant increase of Oct4 transcription, and the reprogramming efficiency was enhanced too (Figure 3K). This synergistic effect supported the idea that Oct4 and Sox2 cooperated in pluripotency induction. We also compared the S-17 MEF reprogramming to traditional reprogramming using overexpressed factors. Unlike S-17 MEFs, the overexpressed factors failed to epigenetically remodel the Sox2 promoter on day 4 (Figure S3J), and no Sox2 transcription from the endogenous loci was effectively detected on days 4 and 12 (Figure 3L). After 3 weeks, overexpressed Oct4 or Sox2 failed to generate any colonies, and overexpression of Oct4, Sox2, and Klf4 (OSK) generated EGFP-positive colonies slightly more than S-17 MEFs with more variation between experiments (Figures 3M and S3K). Previously, we noted that remodeling of both the Oct4 promoter and enhancer is important for pluripotency induction and that targeting the promoter alone is not sufficient for the generation of EGFP-positive colonies (Figures S2A and 2C). Given the fact that key pluripotency factors as well as p300 and the Mediator complex are enriched at the Oct4 distal enhancer in mouse ESCs (Figure 4A), we hypothesize that simultaneous remodeling of the Oct4 promoter and enhancer is required for pluripotency induction. To test that, we used a dual-sgRNA cassette that transcribed two sgRNAs targeting different sites (Figure S4A). The O-127-2066 cassette targets the Oct4 promoter (O-127) and enhancer (O-2066) at a single-cell level. This led to simultaneous remodeling of promoter and enhancer with elevated levels of H3K27ac (Figure 4B). The gene transcription with O-127-2066 was similar to O-127 at days 4 and 8 (Figure 4C). However, after day 8, Oct4 transcription was further elevated in O-127-2066 culture. Particularly, when we replated the cells on days 7 and 11 to allow cell expansion, the overall Oct4 expression in the population dramatically increased (Figure 4C). For O-127 and O-2066 cultures, weak Oct4 expression largely stayed unchanged after day 8 (Figure 4C). Accordingly, by day 12, EGFP-positive colonies were observed in the O-127-2066 culture, and those colonies also expressed Nanog, Sox2, and SSEA-1, indicating the acquired core pluripotency network (Figures 4E and 4F). iPSC lines could be derived from these colonies (Figure 4E). Meanwhile, no colonies were found in the O-127 or O-2066 culture (Figure 4D). The activation of potential off-targets was checked. The top 10 predicted targets for sgRNAs O-127 and O-2066 were examined, and no dramatically activation for off-target genes was seen (Figure S4C). We also tested another two dual sgRNA cassettes O-127-1965 and O-127-2135 in parallel, and similar results were observed (Figure 4D). These data strongly supported that pluripotency was induced by simultaneous remodeling of endogenous Oct4 promoter and enhancer. An authentic pluripotent stem cell line was also achieved. The D-9 line showed similar expression of pluripotency genes to R1 cells and a normal karyotype (Figures 4G and 4H). After injection of D-9 cells into B6-albino blastocysts, live-born chimeric mice were generated at the rate of 60% of the offspring pups (6 out 10) (Figure 4J). This line significantly contributed to the gonadal regions of 75% E13.5 embryos (6 out of 8) (Figure 4I), and germline transmission was confirmed in 50% of male pups (2 out of 4) (Figure 4K). The chromatin remodeling by VP64 is caused by its primary function in recruiting the transcription machinery. To more strictly determine whether epigenetic remodeling is sufficient to initiate reprogramming, we sought to increase the histone acetylation of the Oct4 promoter and enhancer by specific manipulations. Histone acetylation was manipulated because histone H3K27 acetylation synchronously marks the Oct4 promoter and enhancer regions (Figure 4A). p300core only has the acetyltransferase activity domain of p300 and was proved to enhance the targets’ histone acetylation (Hilton et al., 2015Hilton I.B. D’Ippolito A.M. Vockley C.M. Thakore P.I. Crawford G.E. Reddy T.E. Gersbach C.A. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.Nat. Biotechnol. 2015; 33: 510-517Crossref PubMed Scopus (1156) Google Scholar). So we replaced VP64 and generated a dCas9-SunTag-p300core system (Figure S4E). Reprogramming experiments were performed with this dCas9-SunTag-p300core system. We found that p300core culture exhibited similar H3K27ac level to the VP64 counterpart at Oct4 promoter and enhancer, but only 1/30 of the Oct4 transcription was detected in p300core culture at day 5 (Figures S4F and S4G). This can be explained by p300core’s inability to recruit transcription machinery. Cultures were then passaged on days 9 and 14. Interestingly, by day 10, Oct4 levels were comparable in the VP64 and p300core conditions (Figure S4G). Accordingly, EGFP-positive colonies were produced in the p300core cultures, and iPSC lines were generated (Figures S4H and S4I). These observations indicate that the manipulation of histone acetylation with p300core led to chromatin remodeling similar to VP64, although with a noticeable latency in transcriptional activation. Together, our results show that the epigenetic remodeling of Oct4 promoter and enhancer, either through VP64 or p300core, is sufficient to trigger reprogramming toward pluripotency. In this study, we reported that iPSCs were generated with CRISPRa system by targeting single genes, Oct4 or Sox2. The activation of endogenous pluripotent genes had been examined previously with CRISPRa systems, but no iPSCs were established. Several groups succeeded in activating endogenous OCT4 and SOX2 in human 293T cells by targeting the promoter, and murine cells were also tested in some cases (Cheng et al., 2013Cheng A.W. Wang H. Yang H. Shi L. Katz Y. Theunissen T.W. Rangarajan S. Shivalila C.S. Dadon D.B. Jaenisch R. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system.Cell Res. 2013; 23: 1163-1171Crossref PubMed Scopus (547) Google Scholar, Hilton et al., 2015Hilton I.B. D’Ippolito A.M. Vockley C.M. Thakore P.I. Crawford G.E. Reddy T.E. Gersbach C.A. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.Nat. Biotechnol. 2015; 33: 510-517Crossref PubMed Scopus (1156) Google Scholar, Hu et al., 2014Hu J. Lei Y. Wong W.K. Liu S. Lee K.C. He X. You W. Zhou R. Guo J.T. Chen X. et al.Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.Nucleic Acids Res. 2014; 42: 4375-4390Crossref PubMed Scopus (117) Google Scholar, Mali et al., 2013aMali P. Aach J. Stranges P.B. Esvelt K.M. Moosburner M. Ko" @default.
• W2783807303 created "2018-01-26" @default.
• W2783807303 creator A5008419499 @default.
• W2783807303 creator A5023152153 @default.
• W2783807303 creator A5024845093 @default.
• W2783807303 creator A5037788360 @default.
• W2783807303 creator A5059109729 @default.
• W2783807303 date "2018-02-01" @default.
• W2783807303 modified "2023-10-16" @default.
• W2783807303 title "CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency" @default.
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