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

Omnia M. Elsharkasy, Charlotte V. Hegeman, Tom A. P. Driedonks, Xiuming Liang, Ivana Lansweers, Olaf L. Cotugno, Ingmar Y. de Groot, Zoë E. M. N. J. de Wit, Antonio Garcia-Guerra, Niels J. A. Moorman, Sjoerd H. Boonstra, Esmeralda D. C. Bosman, Juliet W. Lefferts, Willemijn S. de Voogt, Jerney J. François, Annet C. W. van Wesel, Samir El Andaloussi, Raymond M. Schiffelers, Sander A. A. Kooijmans, Enrico Mastrobattista, Pieter Vader and Olivier G. de Jong ()
Additional contact information
Omnia M. Elsharkasy: Utrecht University, CDL Research, University Medical Center Utrecht
Charlotte V. Hegeman: Utrecht University, Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences
Tom A. P. Driedonks: Utrecht University, CDL Research, University Medical Center Utrecht
Xiuming Liang: Karolinska Institutet, Division for Biomolecular and Cellular Medicine, Department of Laboratory Medicine
Ivana Lansweers: Utrecht University, Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences
Olaf L. Cotugno: Utrecht University, CDL Research, University Medical Center Utrecht
Ingmar Y. de Groot: Utrecht University, CDL Research, University Medical Center Utrecht
Zoë E. M. N. J. de Wit: Utrecht University, Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences
Antonio Garcia-Guerra: University of Oxford, Department of Paediatrics
Niels J. A. Moorman: Utrecht University, Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences
Sjoerd H. Boonstra: Utrecht University, Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences
Esmeralda D. C. Bosman: Utrecht University, Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences
Juliet W. Lefferts: Utrecht University, Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences
Willemijn S. de Voogt: Utrecht University, CDL Research, University Medical Center Utrecht
Jerney J. François: Utrecht University, CDL Research, University Medical Center Utrecht
Annet C. W. van Wesel: Utrecht University, CDL Research, University Medical Center Utrecht
Samir El Andaloussi: Karolinska Institutet, Division for Biomolecular and Cellular Medicine, Department of Laboratory Medicine
Raymond M. Schiffelers: Utrecht University, CDL Research, University Medical Center Utrecht
Sander A. A. Kooijmans: Utrecht University, CDL Research, University Medical Center Utrecht
Enrico Mastrobattista: Utrecht University, Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences
Pieter Vader: Utrecht University, CDL Research, University Medical Center Utrecht
Olivier G. de Jong: Utrecht University, CDL Research, University Medical Center Utrecht

Nature Communications, 2025, vol. 16, issue 1, 1-18

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-65995-3

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