Fig. 1: Establishment of a CRISPR/Cas9-activated fluorescence reporter platform to study EV-mediated RNA transfer.

a Schematic showing the CRISPR/Cas9-activated fluorescent stoplight reporter system. mCherry is expressed under a CMV promoter, followed by a Cas9-targeted linker region and a stop codon. Two eGFP open reading frames are placed after the stop codon, one or two nucleotides (nt) out of frame, respectively. Upon a Cas9-mediated frameshift in the linker region, either one of these eGFP open reading frames will be permanently expressed alongside mCherry. F2A self-cleaving peptide domains are placed between each fluorescent protein. b Fluorescent microscopy images of stable HEK293T Stoplight⁺spCas9⁺ cells after transfection of a plasmid encoding a sgRNA targeting the linker region of the Stoplight construct (+T sgRNA, bottom row), or a non-targeting sgRNA (+NT sgRNA, top row). Scale bar represents 200 µm. Representative images as observed in three independent experiments. c Flow cytometry analysis of stable HEK293T Stoplight⁺spCas9⁺ cells after addition of PBS, transfection of a non-targeting sgRNA (NT sgRNA), or a sgRNA targeting the Stoplight construct (T sgRNA). Means + SD, n = 3 independent experiments, Student’s t-test. d Cartoon explaining the CROSS-FIRE system. Donor cells (yellow cell, left) express sgRNAs targeting a stoplight construct, which is expressed alongside Cas9 in reporter cells (red cell, middle). Upon functional transfer of sgRNAs from the donor cells to the reporter cell, Cas9 and sgRNA will together activate the stoplight construct in the reporter cell, resulting in permanent eGFP expression (green cell, right), which may then be quantified by fluorescence microscopy or flow cytometry. e, f A five day co-culture of HEK293T Stoplight⁺spCas9⁺ reporter cells with MDA-MB-231 sgRNA⁺ donor cells expressing a targeting sgRNA (T sgRNA), or a non-targeting sgRNA (NT sgRNA), analyzed by fluorescence microscopy (e) and flow cytometry (f). Scale bar represents 200 µm. Representative images as observed in six independent experiments Means + SD, n = 6 independent experiments, Tukey’s multiple comparison test. g Quantification of a five day co-culture of HEK293T Stoplight⁺spCas9⁺ reporter cells with MDA-MB-231 sgRNA⁺ donor cells in varying donor cell: reporter cell ratios by flow cytometry. Means + SD, n = 3 independent experiments, ANOVA. ***p <0.001.