Fig. 5: Simultaneous regulation of transcription and translation with transcription-factor fused, catalytically dead SpCas9.

A Schematic diagram of the simultaneous regulation system. dSpCas9-VPR (catalytically dead SpCas9 fused with a transcriptional activator) was used as the trigger protein. Translational repression and transcriptional activation were monitored with TagRFP and hmAG1, respectively. Tet-responsive elements (TRE) were used as the sgRNA binding site. B Fluorescence microscopy images captured for each condition. Translational repression and transcriptional activation were observed simultaneously when dSpCas9-VPR was transfected. Scale bar, 200 μm. C Quantitative data of reporter expression levels. The data were obtained by imaging analysis. TagBFP was co-transfected as a reference and used to define the transfection-positive area. pSp_gRNA-TagRFP: plasmid for expressing TagRFP, whose translation was regulated by SpCas9. pTRE-hmAG1 plasmid for expressing hmAG1, whose transcription was regulated by Tet-responsive promoter. HEK293FT cells were used in this experiment. TRE: TRE-targeting sgRNA. NT: non-targeting sgRNA. Data are represented as the mean ± SD from 5 independent experiments. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. a.u. arbitrary unit. Source data are provided as a Source Data file.