Fig 1: CRISPR-Sunspot in combination with SunTag activation system for gene activation and mRNA imaging. (A) Lentiviral vector maps for HBG1 activation and mRNA imaging. Workflow of CRISPR-Sunspot system for imaging activated HBG1 mRNA. Stable TRE3G-dCas9-24 × GCN_v4 U2OS cells were infected with scFv-sfGFP-VP64 lentivirus. The cells were then transfected with 2 sets of 3 different sgRNA plasmid with PAMmers targeting HBG1 mRNA for subsequent imaging. (B) Representative images of HBG1 mRNA labeling using the CRISPR-Sunspot system. Scale bars, 20 µm and 5 µm (right panel). The first group (i) showed the representative images of non-activation cell lines (# Ctrl) transfected with the non-targeting control guide RNA with PAMmer. The second group (ii) showed the representative images of non-activation cell lines (# Ctrl) transfected with the sgRNA and PAMmer targeting HBG1 mRNA. The third group (iii) showed the representative images of HBG1 activation cell lines (# 18) transfected with sgRNA and PAMmer targeting HBG1 mRNA. The fourth group (iv) showed the representative images of non-activation cell lines (# Ctrl) transfected by HBG1 overexpression vector and sgRNA with PAMmer targeting HBG1 mRNA. (C) Schematic plots showing the CRISPR-Sunspot system and SunTag-mediated gene activation strategies. (D) SNR of CRISPR-Sunspot labeling of HBG1 mRNA in (B) (n =60, 84, 74, 60 cells). (E) smFISH confirms HBG1 mRNA signals labeling by the CRISPR-Sunspot system in HBG1 activation (by VP64) and HBG1 overexpression (HBG1 OE) U2OS cells. Plots of the arbitrary units (a.u.) along the line indicated the fluorescence intensity in the high-magnification images. Scale bars, 20 µm and 5 µm (right panel). The data are displayed as the mean ± S.E.M. One-way ANOVA with Dunnett's multiple comparisons test was used. *P < 0.05, ***P < 0.001, n.s. not significant.