Design of Polymers for Intracellular Protein and Peptide Delivery

Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.

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.