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. 2019 Jun 28;20(13):3168. doi: 10.3390/ijms20133168

Figure 3.

Figure 3

Application of an MS2 RNA aptamer and additional activators for UBC upregulation, based on the idCas9–VP64 system. (A) Schematic diagram of the working model of the MS2 aptamer and additional activators for the CRISPR activation system. MS2 protein conjugated to C-terminal activator domains, composed of the mouse NF-κB subunit (p65) and human heat shock factor 1 (HSF1), binds to the MS2 RNA stem-loop structure of sgRNA ver. 2. These sgRNA ver. 2 and MS2-p65-HSF1 complexes cooperate with idCas9–VP64 for the upregulation of target genes. (B) HEK293T cells were transiently transfected with idCas9–VP64, MS2-p65-HSF1, and scrambled sgRNA ver. 2 (indicated as -) or sgUBC #1~#4 ver. 2 (alone or in combination), and then treated with 10 μg/mL Dox for 2 days. UBC mRNA levels were determined by qRT-PCR (n = 3 each), normalized to GAPDH, and expressed as the fold change relative to the control (-Dox, scrambled sgRNA ver. 2). (C) Transient transfection of HEK293T cells and Dox treatment were performed as described in (B). Ub conjugates (Ubn) and free Ub were detected by immunoblot analysis using an anti-Ub antibody. α-Tubulin was used as a loading control. (D,F) HEK293T cells were transiently transfected with idCas9–VP64, MS2-p65-HSF1, and scrambled sgRNA (sgScramble) ver. 2 or sgUBC #3 ver. 2, and then treated with 10 μg/mL Dox for up to 4 days. To washout Dox, cells were treated with 10 μg/mL Dox for 2 days and it was then removed from the media for 2 days (2/-2). UBC and UBB mRNA levels were determined by qRT-PCR (n = 3 each), normalized to GAPDH, and expressed as the fold change relative to the control (0 days of Dox treatment, scrambled sgRNA ver. 2). (E) HEK293T cells were transiently transfected with idCas9–VP64, MS2-p65-HSF1, and sgUBC #3 ver. 2, and then treated with 10 μg/mL Dox for up to 4 days. Dox washout (2/-2) was performed as described above. dCas9 mRNA levels were determined by qRT-PCR (n = 3 each), normalized to GAPDH, and expressed as the fold change relative to the control (0 days of Dox treatment, scrambled sgRNA ver. 2). Representative immunoblots are shown from three independent experiments (C). All data are presented as means ± SEM from the indicated number of samples. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control (0 days of Dox treatment or -Dox) or between two groups as indicated by horizontal bars. NS: Not significant.