Fig. 7: Gene activation mediated by SunTag activators in stem cells.

a Schematic illustrating an endogenous Sox-1 reporter in mouse embryonic stem cells for testing the efficiency of CRISPR activation. GFP expression is only detectable upon efficient CRISPR activation. b Quantification of Sox-1 promoter driven GFP expression levels based on fluorescent imaging in single cells. Negative controls (NC) were performed by transfecting VPR without the on-target sgRNA. Each dot represents a single cell (n = 60). All values are mean ± SEM. P values were determined by one-way ANOVA, comparing to the negative control group. c Representative immunofluorescence images of Hoest33342 (blue, nuclear marker) and anti-neurofilament 200 staining (magenta, neuronal marker) in human embryonic stem cells at various conditions. Cells exhibiting neuronal phenotype are highlighted in the white box. Scale bars, 200 μm (original view) and 50 μm (enlarged view). d Quantification of neuronal cells generated by various CRISPRa systems following the use of single or mixed sgRNAs. Data are shown as the mean ± SEM (n = 3 biological replicates). Each dot represents a biological replicate. Dots of the same color indicate that they are from the same batch of experiments. P values were determined by one-way ANOVA, comparing to the negative control (NC, dCas9 + sgNEUROD1) group. e A diagram summarizing the findings of this study. Increasing the number of SunTag-AD arrays (e.g., 3xVPR and 3xVPRF) results in liquid-like condensates and boosts the transcriptional activation of the CRISPRa-SunTag platform. However, when the number of SunTag scaffolds is increased to 10 or more, solid-like condensates form, sequestering co-activators such as p300 and MED1, which exhibit low dynamicity and liquidity, significantly reducing activation strength. We propose that optimal phase separation, reflecting appropriate multivalent interactions within the CRISPRa platform, creates a conducive microenvironment around the transcription start site for efficient activation. Source data are provided as a Source Data file.