Fig. 4: EV-mediated functional delivery of the dCas9-VPR transcriptional activator.

A Schematic of the fluorescent reporter construct for transcriptional activation; pInducer20-eGFP. An eGFP open reading frame is placed after a Tet Responsive Element (TRE) sequence. Transcription of eGFP can either be facilitated by activation of the co-expressed reverse tet-transactivator rTA3 by addition of doxycycline (1) or by introduction of transcriptional activator dCas9-VPR with a sgRNA targeting the TRE sequence (2). Created in BioRender. Utrecht University, P. (2025) https://BioRender.com/v9au9y6. B, C Fluorescence microscopy images (B) and flow cytometry analysis (C) of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 h after addition of doxycycline (0.5 µg/ml), or transfection with plasmids encoding for dCas9-VPR with non-targeting (NT) sgRNAs, targeting (T) sgRNAs, or targeting MS2-sgRNAs. Doxycycline and dCas9-VPR with targeting sgRNAs increase eGFP expression. MFI: mean fluorescence intensity. Scalebar represents 200 μm. Means + SD, n = 3 biologically independent samples, One-way ANOVA with post-hoc Tukey’s multiple comparisons test. D Flow cytometry analysis of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 h after addition of EVs from HEK293T expressing dCas9-VPR alongside either MCP-PhoCl-CD63 or MCP-PhoCl-CD9, in combination with non-targeting (NT), or targeting (T) sgRNAs. Both loading constructs facilitate EV-mediated functional dCas9-VPR delivery, resulting in a significant increase in eGFP mean fluorescence intensity (MFI). 4.0 × 10¹¹ EVs per well. Means + SD, n = 5 independent experiments, One-way ANOVA with post-hoc Dunnett’s multiple comparisons test. * p < 0.05, *** p < 0.001, **** p < 0.0001.