(358h) Intracellular Delivery of Supercharged cas9 RNP’s By Encapsulation in a Polyelectrolyte Complex (PEC) Micelle

Cas9 is the canonical enzyme for gene editing because of its ability to make site-specific edits, deletions, and additions to genomic DNA. However, intracellular delivery of cas9 protein remains a challenge, with many applications relying on viral delivery and integration of the cas9 gene, rather than delivering the protein itself. Here, we present a polymeric delivery system for cas9 ribonucleoprotein complexes (RNPs). Cas9 RNPs were supercharged and encapsulated in PEC micelles. PEC micelles were formed by mixing supercharged cas9 and a hydrophilic-charged diblock copolymer. The micelles were designed to facilitate delivery of cas9 into the cytosol of mammalian cells through endocytosis. The oppositely charged copolymer-supercharged cas9 polyelectrolyte pair electrostatically complexed before micro-phase separating into spherical micelles in a process analogous to bulk phase complex coacervation. The block copolymer and methods of supercharging were chosen to maximize responsivity to endosomal conditions, encouraging micelle dissolution and endosomal escape. Micelles were characterized in terms of salt stability, temporal stability, and responsivity to endosomal pH by dynamic light scattering and transmission electron microscopy. Delivery and localization in mammalian cells were evaluated and the ability to successfully deliver catalytically active cas9 RNP’s to the nucleus was assayed.