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Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26 Page 15

Read out systems for functional delivery of RNA cargo
RNA editing-based assays
Using an inventive assay in which cancer cells were engineered to secrete Cre-recombinase mRNA-
containing EVs to induce Cre-induced RFP to GFP fluorescence conversion in recipient reporter cells,
Zomer et al. showed in vivo cell-cell communication through EVs [127,128] . However, translation of the mRNA
[129]
in EV producer cells may result in co-loading of the mRNA-encoded protein in EVs . To circumvent the
need for mRNA translation in the readout of functional RNA delivery and prevent the possible
contamination with mRNA-encoded protein in EVs, De Jong et al developed a CRISPR/Cas9-based
strategy which they termed CROSS-FIRE (CRISPR-Operated Stoplight System For Functional Intercellular
[126]
RNA Exchange) . In this method, EVs were engineered to contain (non-coding) single guide RNA
(sgRNA) that, upon delivery into Cas9-expressing recipient cells, caused mCherry to GFP fluorescence
conversion by virtue of CRISPR/Cas9-directed frameshifting. The experiments included a 10-day co-culture
of sgRNA-EV producer cells and Cas9-reporter recipient cells, and direct addition of donor EVs to reporter
cells every 72 h for 12 additions with an average dose of 1.1e11 ± 4.9e10 EVs, to facilitate the detection of
changes in GFP fluorescence.

Another RNA-editing-based assay was recently developed to investigate EV-mediated mRNA delivery into
recipient cells, the REMD assay [75,76,121] . EV producer cells were transfected with a Nanoluciferase gene
containing a stop codon, preventing luciferase expression. Recipient cells were made to express CRISPR/
Cas13 and sgRNA targeting the mutated Nluc mRNA. In the case of successful cargo delivery by EVs, the
mutated Nluc mRNA was converted into translationally active mRNA by the presence of CRISPR/Cas13
and sgRNA in the recipient cells, resulting in luciferase expression. In this way, the contamination of EVs
with luciferase protein (as may occur when EV producer cells overexpress non-mutated Nluc mRNA) was
effectively prevented, and luciferase expression in recipient cells rightfully reflected EV-mediated mRNA
delivery.

Of note, the above-mentioned RNA editing-based assays are useful for quantifying EV-mediated RNA
delivery [Table 2], but are not capable of pinpointing the subcellular sites of cargo delivery. Therefore, a
combination of techniques is needed to correlate EV uptake, intracellular trafficking and escape of EV cargo
from endosomes with the induction of phenotypic changes in recipient cells, i.e., functional delivery of EV
cargo.

Controls in cargo delivery assays
For functional cargo delivery assays, it would be helpful to include a dose curve to determine if an increase
in the functional outcome correlates with an increase in the added amount of EVs. The amount of EVs
should be based not only on the total protein content but also on the number of particles, e.g., measured by
Nanoparticle Tracking Analysis (NTA), because contaminants can lead to an overestimation of the EV
[130]
protein content . EVs should be used only after thorough characterization following MISEV(2018)
guidelines to enable the reproducibility of experiments among different labs. Briefly, markers typically
enriched in EVs (CD9, CD81, TSG101, ALIX) and the absence of markers for specific organelles
(endoplasmic reticulum, Golgi) should be demonstrated. A morphological investigation should be
performed with electron microscopy. Further EV purification after ultracentrifugation should be done with
sophisticated techniques such as sucrose gradient centrifugation, ultrafiltration, or affinity chromatography.
The choice of a specific technique depends on the cargo being loaded in the EVs. For miRNA, siRNA, or
mRNAs, it is indispensable to remove protein-nucleic acid aggregates that may co-precipitate with EVs in
ultracentrifugation and can be separated, e.g., with sucrose gradient centrifugation.