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. 2018 Jul 6;8:10286. doi: 10.1038/s41598-018-28599-0

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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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I-CreI in vitro cleavage and binding experiments. (a) In vitro cleavage titration analysis for I-CreI_D75N_target and I-CreI_3115_target using linearized plasmids containing the cleavable target sequences. (b) Validating 5NNN non-cleavable target patterns using labelled duplex DNA targets. Lanes: 1, I-CreI_D75N_target; 2, I-CreI_D75N_target in the presence of I-CreI_D75N protein; 3, I-CreI_3115_target in the presence of I-CreI_3115 protein; 4, I-CreI_3115_target; 5, I-CreI_D75N_target-null (non-cleavable derivative); 6, I-CreI_D75N_target-null in the presence of I-CreI_D75N protein; 7, I-CreI_3115_target-null (non-cleavable derivative) in the presence of I-CreI_3115 protein; 8, I-CreI_3115_target-null (non-cleavable derivative). (c) I-CreI_D75N/I-CreI_D75N_target or target-null binding by microscale thermophoresis (in the presence of 10 mM CaCl₂). (d) I-CreI_3115/I-CreI_3115_target or target-null binding by microscale thermophoresis (in the presence of 10 mM CaCl₂). The change in thermophoresis is plotted against the concentration of DNA in a logarithmic scale and fitted to a 1:1 binding model to yield the corresponding K_Ds. Numbers in brackets represent the 68.3% confidence interval calculated by ESP (error-surface projection).