Figure 4.

Engineered EVs efficiently delivered Cre recombinase mRNA in vitro. a) Illustration of triple co‐transfection methodology for Cre‐PUFe mRNA EV production from HEK293T producer cells, and subsequent EV production. Figure created using BioRender. b) Average particle size determination by NTA of Cre‐PUFe mRNA EVs and control EVs with VSVg co‐expression. c) In vitro uptake analysis of T47D Traffic Light Cre reporter cells treated with equal amounts of Cre‐PUFe mRNA EVs at a dose of 2 pg and 20 pg mRNA per 1 × 10⁴ cells, or particle count‐matched control EVs, both with VSVg expression. Frequencies of genomically edited Cre reporter cells were assessed at 24, 48, and 72 h post treatment by flow cytometry. Cells treated with mRNA loaded EVs show a time‐ and dose‐dependent increase in genomically edited cell frequencies, even exceeding efficiencies achieved by Cre plasmid transfection (black). Data presented as single data points and mean (column), n = 4; P‐values were calculated by two‐way ANOVA using Šìdàks Multiple Comparisons test; α = 0.05; ns (non‐significant) p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.