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. 2019 Oct 2;9:14219. doi: 10.1038/s41598-019-50733-9

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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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CRISPR Knock-In CXCR4 clones allow for accurate single molecule cluster analysis compared to transfected cells. (A) Single molecule imaging of HaloTag CXCR4 Knock-Ins allows for effective single molecule localisation microscopy through labelling with Janelia Fluor 549, and subsequent cluster analysis using persistence based clustering. (B) CXCR4 knock-in and Hek293T transfected with a CXCR4-HaloTag were subject to single molecule imaging and cluster analysis after treatment with CXCL12 (10μM). CRISPR knock-ins demonstrate a significant increase in median cluster area on ligand treatment (*p = 0.0272), while transfected cells show no significant change. (C) Histograms showing percentage of clusters across a range of areas show that while a substantial shift towards larger cluster sizes is evident in CRISPR knock-ins, this is reduced in transfected cells. (D) The percentage of clusters below 50 nm in radius is significantly reduced in CRISPR cells (*p = 0.0384) upon treatment with CXCL12 (upper panel), with a parallel increase in the percentage of clusters of larger sizes consistent with endocytic trafficking observed (*p = 0.0133) (lower panel). Conversely, no significant change in either collection of cluster sizes is evident in transfected cells. (n = 3, S.E.M, paired t-test).