Fig. 1: CRISPRa systems modulates transcriptional bursts.

a Schematics illustrating the design of different CRISPRa systems. b Time-lapse images depicting transcriptional bursts of the miniCMV-TriTag^mTagBFP reporter induced by various CRISPRa systems. Arrows highlight nascent RNAs labeled with stdMCP-tdTomato at transcription sites. Each cell was imaged for 2 h with 2-min intervals. Only images taken between 30 to 84 min are displayed. Scale bars, 5 μm. c Representative traces (red) demonstrating the kinetics of transcriptional bursting at miniCMV loci induced by various CRISPRa systems. Gray traces indicate the intensity of the background signal. d Bar graph presenting the average durations of bursts and pauses induced by CRISPRa. From left to right, the burst numbers are 117, 150, 217, 103, 140, 119, and 106, while the pause numbers are 123, 93, 237, 49, 134, 99 and 95, respectively. Data are shown as mean ± SEM. e Burst amplitude determined by measuring the maximum total intensity of stdMCP-tdTomato spots within each burst. From left to right, cell numbers are 54, 89, 128, 135, 115, 111 and 115. Data are shown as mean ± SEM. P values were calculated by one-way ANOVA test, comparing to the VP64 group. f Fluorescent imaging-based quantification of BFP expression levels driven by miniCMV following CRISPR activation for 48 h. n = 100 cells. Data are shown as mean ± SEM. P values were calculated by one-way ANOVA test, with the negative control (NC) as the reference group. g Flow cytometry analysis of BFP expression levels to determine the activation ratio of individual CRISPRa systems. Transfection-positive cells were gated based on an internal fluorescent reporter. Data are shown as mean ± SEM. n = 3 independent experiments. P values were calculated by one-way ANOVA test, comparing to the NC group. In (f) and (g), negative controls (NC) were conducted by transfecting dCas9 lacking AD fusions and sgRNA targeting miniCMV. Source data are provided as a Source Data file.