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. 2018 Dec 24;30(3):667–678. doi: 10.1021/acs.bioconjchem.8b00856

Figure 7.

Figure 7

ECO mediated GFP silence with the CRISPR/Cas9 system. (A) In vitro transfection of ECO/psgRNA and ECO/pCas9 (particle ratio 1:1) in NIH3T3-GFP cells, with the dose of each plasmid at 1, 1.5, 2, and 2.5 μg/well in the transfection media. Cas9 expression (blue), gRNA expression (red), and GFP knockdown (green) were imaged by confocal microscopy. (B) Quantitative flow cytometry measurements of GFP knockdown efficiency represented the percentage of GFP fluorescent intensity normalized to nontreated control. (C) GFP knockdown efficiency in mRNA levels measured by qRT-PCR. Cells were treated with ECO/CRISPR/Cas9 nanoparticles at a dose of 2 μg/well and mRNA levels were evaluated at 24 and 48 h. (D) Quantitative flow cytometry measurements of sgRNA expression represented by percentage of cells with mCherry expression. (E) Western blot showing Cas9 protein expression 72 h after CRISPR/Cas9 treatments by ECO at a dose of 2 μg/well. (NS = nonspecific control, β-actin expression as inner control). (Error bars = ± std. * p < 0.05 relative to untreated control). Scale bars = 200 μm.