Journal: bioRxiv

Article Title: A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

doi: 10.1101/2024.05.24.595612

Figure Lengend Snippet: 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). b,c Fluorescence microscopy images (b) and flow cytometry analysis (c) of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 hours 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. Doxycyline and dCas9-VPR with targeting sgRNAs increase eGFP expression. MFI: mean fluorescence intensity. Scalebar represents 200 μm. Means + SD, n = 3, One-way ANOVA with post-hoc Dunnett’s multiple comparison test. d, Flow cytometry analysis of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 hours 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). 4.0x10 11 EVs per well. Means + SD, n = 5, One-way ANOVA with post-hoc Dunnett’s multiple comparison test. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the generation of a lentiviral doxycycline-inducible eGFP expression construct, an eGFP open reading frame was transferred from pDONR221- eGFP (Addgene #25899) into pInducer20 (Addgene #44012) using the Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Construct, Activation Assay, Sequencing, Fluorescence, Microscopy, Flow Cytometry, Expressing, Transfection, Comparison, Functional Assay