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. 2025 Dec 11;17:751. doi: 10.1038/s41467-025-67438-5

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© The Author(s) 2025

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — (a) Illustration of the dCas9-SunTag blockade system. 38 binding sites (BS) were inserted downstream of the Sis element to provide multiple binding sites for the dCas9-SunTag system. (b) Western blot confirmation of dCas9 expression in the dCas9-SunTag blockade line, derived from the Wapl-AID2 CTCF-Nm line. The experiment was repeated twice with similar results. (c) Reverse transcription-quantitative PCR (RT-qPCR) analysis showing the fold enrichment of gRNA expression in the dCas9-SunTag blockade line, relative to the control BS-only line. Data are shown as mean ± SD from three independent replicates. (d) ChIP-qPCR analysis showing the fold enrichment of dCas9-SunTag binding at the BS in the dCas9-SunTag blockade line and the control BS-only line. A region near Igκ-RS was used as a negative control region. Data are shown as mean ± SD from three independent replicates. (e) Left, 3C-HTGTS profiles demonstrating formation of loop barrier near dCas9 binding sites in the dCas9-SunTag blockade line. Right, Quantification of bait interactions. Libraries were size-normalized, and interaction signals were counted for the indicated regions. Data are shown as mean from two independent replicates. (f) Left, HTGTS-V(D)J-Seq analysis of Vκ usage frequency (%) in the BS-only line (top) and the dCas9-SunTag blockade line (bottom). Right, summary of deletional and inversional Vκ usage in proximal versus middle/distal Vκ regions in the BS-only and dCas9-SunTag blockade lines. Data are shown as mean ± SD from three and four independent replicates, respectively. P values were calculated using unpaired, two-tailed Student’s t test.