Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain

Abstract

Genome editing using CRISPR/Cas9 has attracted considerable attention for the treatment of genetic disorders and viral infections. Co-delivery of Cas9 mRNA and single guide (sg)RNA is a promising strategy to efficiently edit the genome of various cell types, including non-dividing cells, with minimal safety concerns. However, co-delivery of two RNA species with significantly different sizes, such as Cas9 mRNA (4.5 kb) and sgRNA (0.1 kb), is still challenging, especially in vivo. Here, we addressed this issue by using a PEGylated polyplex micelle (PM) condensing the RNA in its core. PM loading sgRNA alone released sgRNA at minimal dilution in buffer, while PM loading Cas9 mRNA alone was stable even at higher dilutions. Interestingly, co-encapsulating sgRNA with Cas9 mRNA in a single PM prevented sgRNA release upon dilution, which led to the enhanced tolerability of sgRNA against enzymatic degradation. Subsequently, PM with co-encapsulated RNA widely induced genome editing in parenchymal cells in the mouse brain, including neurons, astrocytes, and microglia, following intraparenchymal injection, at higher efficiency than that by co-delivery of PMs loaded with either Cas9 mRNA or sgRNA separately. To the best of our knowledge, this is the first report demonstrating the utility of RNA-based delivery of CRISPR/Cas9 in inducing genome editing in the brain parenchymal cells. Furthermore, the efficiency of genome editing using PMs was higher than using a non-PEGylated polyplex, due to the enhanced diffusion of PMs in the brain tissue. The results reported herein demonstrate the potential of using PMs to co-encapsulate Cas9 mRNA and sgRNA for in vivo genome editing.