Altogether, this work further broadens the utility of EVs for (bio)therapeutic delivery strategies and underlines the potential for EV-mediated treatment of genetic diseases.

As there is no direct fusion to Cas9, one can readily swap out Cas9-based moieties, sgRNAs, and EV-enriched loading proteins. We compare various EV-enriched proteins, and show delivery of multiple Cas9-based moieties: spCas9, transcriptional activator dCas9-VPR and adenine base editor ABE8e.

Here, we combine 2 strategies: active Cas9 loading into EVs by fusing MS2 Coat Protein (MCP) domains to EV-enriched proteins which bind to MS2 hairpins in the spCas9 sgRNA tetraloop and/or the 2nd stemloop, alongside regulated cargo release from the EV membrane using UV-cleavable PhoCl domains.

This work was done in collaboration with Zhiyong Lei from the University Medical Center Utrecht and demonstrates the potential for lipopeptide-mediated Cas9 delivery, both in vitro and in vivo.

Furthermore we show that C18:1-LAH5 CPPs are suitable for in vivo Cas9 RNP intramuscular delivery in a murine Ai9 reporter model, demonstrating gene editing in both endogenous and fluorescent reporter genes.

Testing various fatty acid tail modifications, we show that N-terminal oleic acid (C18:1) modification of LAH5 results in increased stability, Cas9 RNP delivery, and both Cas9 gene editing (NHEJ) and gene repair (HDR) in various reporter cell lines.

As a follow-up on previous work where we showed efficient in vitro delivery of Cas9 RNP using the amphipathic LAH5 CPP in various cell lines we aimed to to further increase nanocomplex stability and Cas9 delivery in serum-rich conditions by incorporation of fatty-acid modifications.

In this manuscript, performed in collaboration with Zhiyong Lei at the UMC Utrecht, we improve the cell-penetrating peptide (CPP)-mediated delivery of Cas9 RNP by N-terminal incorporation of various fatty acids.

I'd like to be added please!