Fig. 1: dCas9-VP64-IDR robustly activates reporter gene with high specificity.

a Schematic representation of dCas9-VP64-IDR targeting a Tet-on GFP reporter. The GFP expression in this reporter is regulated by seven tandem tetracycline operator (7xTetO) sequences along with a minimal CMV promoter. Binding of the dCas9-VP64-IDR fusion protein to the TetO sites activates GFP expression. b Domain architectures of proteins containing intrinsically disordered regions (IDRs). The specific IDR fragments fused to dCas9-VP64 in this study are highlighted in yellow. c Boxplot illustrating the GFP intensity in HEK293R cells expressing dCas9-VP64 or dCas9-VP64-IDR fusions, in combination with a guide RNA targeting the TetO elements (gTetO). The results are presented as the median GFP intensity, along with the 25th and 75th quartiles, as well as the 5th and 95th percentiles, and are representative of three independent experiments. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Cell numbers from left to right (n = 14,983, 13,741, 15,366, 14,226, 18,070, 16,249, 14,173, 12,839, 12,335, 9112, 16,921, 19,733, 12,970). d Volcano plot comparing the gene expression profile of HEK293R cells expressing dCas9-VP64-FUS with a gRNA targeting TetO (gTetO) to that with a scrambled gRNA (gScr) control. The x-axis represents the Log2 fold change in gene expression between the two conditions, while the y-axis displays −Log10 p value. Differentially expressed genes were determined using the criteria of |Log2 (fold change)| > 1 and FDR < 0.05, with GFP (marked in red) being the only gene meeting these criteria. Statistical significance was determined by two-sided Wald test with Benjamini–Hochberg correction. e Correlation of gene expression profile of HEK293R cells expressing dCas9-VP64-FUS with a gRNA targeting TetO (gTetO) to that with a scrambled gRNA (gScr) control. The x-axis represents the Log2 TPM of control group and y-axis displays the Log2 TPM of activation group. Pearson correlation coefficient (R) = 0.99. The source data for Fig. 1d, e are deposited under accession number GSE248523. Source data are provided as a Source data file.