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. 2018 May 9;8:7424. doi: 10.1038/s41598-018-25454-0

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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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Interphase Organization and Dynamics of RNA Pol I. (a) A homologous donor vector containing the Dendra2 fluorescence protein flanked by two 500 bp homologous arms. When co-transfected with a plasmid expressing Cas9 along with a targeted sgRNA, homology directed repair at the protospacer adjacent motif (PAM) cut site induces insertion of the Dendra2 sequence onto the N-terminus of the RPA194, the largest subunit of RNA Pol I. We imaged a total of 77 cells on seven separate days. (b) Bright-field and conventional fluorescence imaging of the pre-converted state of Dendra2-RPA194 in a U2OS cell line. The nucleus is demarcated with a dashed line while the contours of the nucleoli are demarcated with a solid white line. The polymerase appears to cluster in distinct foci within the nucleoli. (c) Super-resolution reconstruction of the Dendra2-RPA194. At the super-resolution level, foci remain visible. (d) A sample time trace of the cluster marked in yellow in the super-resolution image. In the cumulative trace, the detections show an initial linear slope indicating that the cluster was pre-existing and slowly level off suggesting that the cluster is stable.