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. 2017 May 19;45(13):e118. doi: 10.1093/nar/gkx309

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© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — Construction of miR-Cas9-ON switch. (A) Mechanism of the ON system. The expressions of L7Ae from mRNA2 and Cas9 protein from mRNA1 are suppressed by target miRNA (here, miR-21) and L7Ae, respectively. L7Ae suppresses the translation of Cas9 protein by interacting with its binding motif (K-turn), and miR-21 suppresses the translation of L7Ae by interacting with its anti-sense region, resulting in Cas9 translation. (B) Flow cytometry histograms of EGFP expression. The x- and y-axes indicate EGFP intensity and cell number, respectively. An increase of the EGFP negative population was observed in miR-21-responsive L7Ae mRNA (ON) compared with control L7Ae mRNA (OFF). This experiment was repeated three times and representative histograms are shown. Control L7Ae mRNA (L7Ae mRNA without miRNA-responsive element). Constitutive ON (without L7Ae mRNA). (C) Quantification of relative Cas9 activity from (B). An increase of the EGFP negative population was observed in miR-21-responsive L7Ae mRNA (ON) compared with control L7Ae mRNA (OFF). Each Cas9 activity was normalized by Constitutive ON (- L7Ae). Control L7Ae mRNA (L7Ae mRNA without miRNA-responsive element). Constitutive ON (without L7Ae mRNA). Negative control is Cas9–sgRNA_DMD that targets DMD gene. Error bars indicate the mean ± SD (n = 3).