Delivery technologies for the CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing1. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles (typically >100 nm diameter); however, such systems are limited by variability in the loading of the cargo. Here, we report the design of customizable synthetic nanoparticles for the delivery of Cas9 nuclease and a single-guide RNA (sgRNA) that enables the controlled stoichiometry of CRISPR components and limits the possible safety concerns in vivo. We describe the synthesis of a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating, called a nanocapsule (NC), around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. The NC is synthesized by in situ polymerization, has a hydrodynamic diameter of 25 nm and can be customized via facile surface modification. NCs efficiently generate targeted gene edits in vitro without any apparent cytotoxicity. Furthermore, NCs produce robust gene editing in vivo in murine retinal pigment epithelium (RPE) tissue and skeletal muscle after local administration. This customizable NC nanoplatform efficiently delivers CRISPR RNP complexes for in vitro and in vivo somatic gene editing.
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The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.
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We thank the James Thomson lab for the use of their BD FACSCanto II, and the University of Wisconsin Biotechnology Center for providing facilities and services. We thank Aldevron for supplying reagents and technical support. We are grateful to Q. Chang who provided us with the Ai14 tdTomato transgenic mice used for the initial evaluation. We acknowledge the generous financial support from the National Institute for Health (1-UG3-NS-111688-01, R01EY024995, 1R35GM119644, R01NS091540, R01HL143469 and R01 HL129785), the National Science Foundation (CBET-1350178 and CBET-1645123), the Wisconsin Alumni Research Foundation and the Wisconsin Institute for Discovery. The authors also acknowledge financial support from the University of Wisconsin–Madison.
G.C., A.A.A., Y.W., R.X., K.S. and S.G. have filed a patent application on this work.
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