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. 2020 Jun 26;11:3232. doi: 10.1038/s41467-020-17029-3

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© The Author(s) 2020

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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Fig. 2 — a T7EI cleavage assay of DNA isolated from HeLa-Luc cells treated with various formulations. Highly effective gene editing was mediated by 5A2-DOT-10 delivering Cas9/sgLuc RNPs (1/3 and 1/5). Red arrows indicate cleavage bands. Indels (%) at Luc locus was quantified by Sanger sequencing and TIDE analysis. This experiment was repeated three times independently with similar results. b Fluorescence microscopy images of HeLa-GFP cells after treatment with various formulations (n = 3 biologically independent samples). Scale bar = 100 μm. 5A2-DOT-10 Cas9/sgGFP treatment significantly decreased GFP fluorescence. c Flow cytometry analysis of HeLa-GFP cells after treatment with various formulations. The peak of GFP-positive cells shifted completely to the left only for the 5A2-DOT-10 Cas9/sgGFP group, indicating almost all GFP-positive cells went dark. d Time-dependent GFP fluorescence intensity of HeLa-GFP cells after various treatments (mean ± s.e.m., n = 3 biologically independent samples). Permanent GFP fluorescence loss was observed with 5A2-DOT-10 Cas9/sgGFP treatment, which was supported by Sanger sequencing and TIDE analysis. e 5A2-DOT-10 Cas9/sgGFP LNPs were stored at 4 °C for 2 months. The nanoparticle diameter and PDI was monitored over time (mean ± s.e.m., n = 4 biologically independent samples). f Periodic treatment of HeLa-GFP cells with stored LNPs showed that no activity was lost, indicating long-term LNP and RNP stability (mean ± s.e.m., n = 3 biologically independent samples). g Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9/sgGFP alone, 5A2-DOT-10, C12-200-DOT-10, MC3-DOT-10, C12-200 LNPs, MC3 LNPs, and Cas9/sgGFP-loaded RNAiMAX (mean ± s.e.m., n = 3 biologically independent samples). The GFP fluorescence significantly decreased after treated with all three DOTAP-modified formulations. TIDE analysis of Sanger sequencing data further confirmed the highest gene editing efficiency was with 5A2-DOT-10 LNPs. h Mean fluorescence intensity (%) of Hela-GFP cells after treatment with 5A2-DOT-10 formulated with Cas9/sgGFP in different buffers. Neutral buffer was required for RNP encapsulation and delivery (mean ± s.e.m., n = 3 biologically independent samples). One-way ANOVA followed by Dunnett’s multiple comparison test was used to determine the significance in g and h. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Source data are in the Source Data file.