A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains one of the major hurdles in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles released by cells, that play an important role in intercellular communication. Due to their innate capability of intercellular transfer of proteins, RNA, and various other biological cargos, EVs have emerged as a novel promising strategy for the delivery of macromolecular biotherapeutics, including CRISPR-Cas9 ribonucleoproteins. Here, we present a versatile, modular strategy for the loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins (MCPs) fused to EV-enriched proteins to MS2 aptamers incorporated into single guide RNAs (sgRNAs), in combination with a UV-activated photocleavable linker domain, PhoCl. Combined with the Vesicular stomatitis virus G (VSV-G) protein this modular platform enables efficient loading and subsequent delivery of the Cas9 ribonucleoprotein complex, which shows critical dependence on the incorporation and activation of the photocleavable linker domain. As this approach does not require any direct fusion of Cas9 to EV-enriched proteins, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e, as confirmed by various sensitive fluorescent reporter assays. Taken together, we describe a robust and modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering as well as transcriptional regulation, underlining the potential of EV-mediated strategies for the treatment of genetic diseases. ### Competing Interest Statement P.V. is consultant and S.E.A. is consultant and stakeholder in Evox Therapeutics Limited, Oxford, United Kingdom.