A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release
Publication date
2025-11-21
Authors
Elsharkasy, Omnia M
Hegeman, Charlotte V
Driedonks, Tom A P
Liang, Xiuming
Lansweers, Ivana
Cotugno, Olaf L
de Groot, Ingmar Y
de Wit, Zoë E M N J
Garcia-Guerra, Antonio
Moorman, Niels J A
Editors
Advisors
Supervisors
Document Type
Article
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License
cc_by
Abstract
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 a major hurdle in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles that play an important role in intercellular communication, and have an innate capability of intercellular transfer of biological cargos, including proteins and RNA. Here, we present a versatile, modular strategy for EV-mediated loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins fused to EV-enriched proteins to MS2 aptamers incorporated into guide RNAs, in combination with a UV-activated photocleavable linker domain, PhoCl. Moreover, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e. Taken together, we describe a robust, modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering and transcriptional regulation.
Keywords
Aptamers, Nucleotide/metabolism, CRISPR-Associated Protein 9/metabolism, CRISPR-Cas Systems/genetics, Extracellular Vesicles/metabolism, Gene Editing/methods, HEK293 Cells, Humans, RNA, Guide, CRISPR-Cas Systems/genetics, Ultraviolet Rays
Citation
Elsharkasy, O M, Hegeman, C V, Driedonks, T A P, Liang, X, Lansweers, I, Cotugno, O L, de Groot, I Y, de Wit, Z E M N J, Garcia-Guerra, A, Moorman, N J A, Boonstra, S H, Bosman, E D C, Lefferts, J W, de Voogt, W S, François, J J, van Wesel, A C W, El Andaloussi, S, Schiffelers, R M, Kooijmans, S A A, Mastrobattista, E, Vader, P & de Jong, O G 2025, 'A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release', Nature Communications, vol. 16, no. 1, 10309. https://doi.org/10.1038/s41467-025-65995-3