A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing

Abstract

Delivery technologies for the CRISPR-Cas9 gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing ¹ . 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 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), enabling controlled stoichiometry of CRISPR components and limiting 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 pre-assembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and a sgRNA. The NC is synthesized by acrylate-based 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 following local administration. This customizable NC nanoplatform efficiently delivers CRISPR RNP complexes for in vitro and in vivo somatic gene editing.