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. 2020 Jan 20;10:635. doi: 10.1038/s41598-020-57500-1

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© The Author(s) 2020

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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Generation and validation of AAVS1-integrated inducible dCas9-KRAB and dCas9-VPR hPSC lines. (a) Schematic overview of AAVS1 targeting strategy in H1 hPSCs with TRE3G-driven dCas9-KRAB (left) or dCas9-VPR (right) cassettes and TALENs that target AAVS1 and confer G418 resistance upon on-target integration. (b) Quantification of flow cytometry analysis of EGFP fluorescence in dCas9-KRAB cells after 48 hours of doxycycline treatment (+48 h) followed by removal of doxycycline for 120 (−120 h) and 168 hours (−168 h) in comparison to no GFP control H1 cells (control). (c) Quantification of flow cytometry analysis of EGFP fluorescence in dCas9-VPR cells after 48 hours of doxycycline treatment (+48 h) followed by removal of doxycycline for 120 (−120 h) and 168 hours (−168 h) in comparison to no GFP control H1 cells (control). (d) dCas9-KRAB protein expression in absence of doxycycline (−Dox), after 24 and 48 hours doxycycline treatment (+24 h, +48 h), and after washout of doxycycline for 24, 72, 120, and 168 hours (−24 h, −72 h, −120 h, −168 h). (e) dCas9-VPR protein expression before doxycycline treatment (-Dox), after 24 and 48 hours doxycycline treatment (+24 h, +48 h) and after washout of doxycycline for 24, 72, 120, and 168 hours (−24 h, −72 h, −120 h, −168 h). Images in d,e derived from Wes analysis (ProteinSimple).