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. 2017 Nov 23;7:16174. doi: 10.1038/s41598-017-16279-4

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© The Author(s) 2017

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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Sustained CRISPR/Cas9 mediated silencing of luc2 in vivo. (a) The short guide RNA (sgRNA) specific for luc2 was designed to target the 5′ region of the gene, to increase the likelihood of inducing a frame-shifting deletion that would knock out luciferase activity by generating a premature termination codon. (b) An in vitro dual-luciferase assay demonstrated successful targeting of luc2 by the sgLuc2 construct, as shown by a significant reduction in luciferase activity when normalized to untreated cells (data normalised against the untreated control = 100%). (c) Representative image of mice displaying a maximal reduction in luc2 expression after injection with the sgLuc2 construct (right eye). This image was taken from the mouse represented by the green line in panel (d), below, at 7 days post treatment. (d) After treatment, the corneal luciferase activity of each mouse was quantified using a Xenogen IVIS live animal imager every day for 7 days, then every 7 days thereafter, for a total of 6 weeks. Luciferase activity for each treatment group expressed as a percentage of control (R/L ratio %).