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. 2018 Nov 26;8:17348. doi: 10.1038/s41598-018-35587-x

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© The Author(s) 2018

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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CRISPR-on modulation of GJB6 and non-coding lncRNA RP11-264J4.10 expression via a putative enhancer. (a) UCSC browser snapshot presenting the candidate enhancer lifted over from mouse (mm10) to human (hg19) (track: “P22 lifted LMRs”, black box indicators); additional information derived from ENCODE regulation hub is presented to support the characterization of the candidate sequence as an enhancer (track: ‘transcription’ - purple signal = NHEK cell line, track: ‘NHEK ChromHMM’ – orange indicating ‘Active enhancer’). Below, zoomed view of the candidate sequence, with the location of the lifted-over LMR, gRNA targets and ATOH1 TFBS indicated. (b, c) Expression of GJB2, GJB6 and ATOH1 (b) and RP11-264J4.10 ncRNA (c) measured by qRT-PCR (normalized to GAPDH, compared to ‘no DNA’, n = 4–5, ***p < 0.01, *p < 0.05). (d) ATOH1 transcription factor binding motif found at the candidate enhancer, score is calculated by JASPAR. (e) Suggested mechanism of GJB6 regulation by the putative enhancer, shown in two models. Model 1 depicts a physical distal interaction between the GJB2 proximal putative enhancer (yellow box) and the GJB6 promoter (light orange box) mediated by ATOH1 and/or the lncRNA (purple). Model 2 demonstrates the folding of the chromatin, linking the putative enhancer with the GJB6 promoter, mediated by ATOH1 and/or the lncRNA.