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. 2019 May 9;10:2119. doi: 10.1038/s41467-019-10146-8

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Methodological variance is not the source of unresponsiveness to trans-activation. a dCas9 Protein levels do not differ between populations. Sox1^GFP-positive and -negative cells were sorted from control and activated populations (S1-9), and an immunoblot for dCas9 was performed. dCas9 levels did not significantly vary between the respective samples; Image was cropped for clarity. b Resistance to activation is gene-dependent. Actc1 gRNAs (A1-9) and Sox1 gRNAs (S1-9) were co-transduced into NPCs and immunostained for the target proteins after 7 days. While almost all cells induced Actc1 expression, only a small subpopulation was able to activate the Sox1 signal. Dashed yellow lines mark magnified areas; scale bar: 100 µm. c dCas9 binding does not vary between Sox1-positive and -negative NPCs. Sox1^GFP-positive and -negative cells were sorted from the activated population (SoxProm), and ChIP for dCas9 was performed at the Sox1 locus as well as on the promoters of Gapdh, Actc1, and Oct4 (serving as negative controls); dCas9 is strongly enriched at the Sox1 locus; no significant difference in DNA binding between responsive and unresponsive NPCs was detected. Data depict one of three biological replicates performed on different days in different clonal lines., mean and standard error of the mean of n = 3 technical replicates shown; n.s., not significant. d dCas9-VPR does not overcome the heterogeneity of gene activation observed with dCas9-VP64. NPCs stably expressing Sox1 targeting gRNAs (S1-9) were transfected with either dCas9 only, dCas9-VP64 or dCas9-VPR. Although higher Sox1 mRNA levels were seen when gene induction was conducted with dCas9-VPR, no significant difference in the number of Sox1^GFP-positive cells was detected between the dCas9-VP64- and dCas9-VPR-expressing NPCs (data depict one of three biological replicates, mean and standard error of the mean of n = 3 technical replicates shown)