Fig. 4: Osmotic pressure regulates transcription of target genes with dCas9-SunTag system.
From: Osmotic pressure regulates DNA labelling and transcription with dCas9-SunTag system in live cells
a Schematic of the transcription activation of a target gene with dCas9-SunTag-FP-AD. AD, activation domain. b Relative mRNA expression of HBG1 with the dCas9-SunTag recruiting the transcription activation domains VPR or VP64 under different osmotic conditions. Statistics for biological replicates with n = 4 for H2O and n = 6 for PEG. c Schematic of CRISPRTag-TRE-luciferase-24×MS2 DNA locus labelled and activated by dCas9-SunTag-FP-AD, with nascent MS2-tagged luciferase mRNA labelled by MCP-mCherry. d Relative luciferase expression with the dCas9-SunTag-FP-AD under different osmotic conditions. n = 3 independent experiments. e Left, snapshot showing the CRISPRTag-TRE-luciferase-24×MS2 locus labelled with dCas9-SunTag-sfGFP-AD. From 3D orthogonal view along z axis. Scale bar, 5 μm. Right, enlarged 2D snapshots at the target locus (marked by a white square on the left image) over time, indicating the transcription burst. Scale bar, 500 nm. 2D radial distributions of sfGFP and mCherry intensity are shown at the bottom. f Frequency of mRNA burst under various osmotic treatments. Statistics for target loci over 20 min. VPR, ctrl, n = 8; 20% H2O, n = 4; 10% PEG, n = 4. VP64, ctrl, n = 10; 20% H2O, n = 7; 10% PEG, n = 3. All statistics above are presented as mean ± SE.
