Figure 1.

Engineering strategy for endogenous mRNA loading into EVs. a) EV sorting strategy: four novel RNA‐binding domains (RBD), the non‐cleaving mutant Cas6f and three engineered versions of the designer Pumilio and FBF homology domain (PUF), termed PUFm, PUFe, and PUFx2, were fused via a glycine‐rich linker peptide to the C‐terminus of the EV sorting protein CD63. As control for passive loading (non‐binding control, NBC), CD63 was fused to the MS2 coat protein (MCP, as published by Hung et al., 2016). b) mRNA‐binding strategy: mRNA coding sequences were codon‐optimized and engineered to contain 6–10 repeats of the specific high‐affinity motif recognized by the respective RNA‐binding domain in their 3′UTR. Figure was created using BioRender.