Genome Editing in a Wide Area of the Brain Using Dendrimer-Based Ternary Polyplexes of Cas9 Ribonucleoprotein.

A preassembled Cas9/single-guide RNA complex (Cas9 ribonucleoprotein; Cas9 RNP) induces genome editing efficiently, with small off-target effects compared with the conventional techniques, such as plasmid DNA and mRNA systems. However, penetration of Cas9 RNP through the cell membrane is low. In particular, the incorporation of Cas9 RNP into neurons and the brain is challenging. In the present study, we have reported the use of a dendrimer (generation 3; G3)/glucuronylglucosyl-β-cyclodextrin conjugate (GUG-β-CDE (G3)) as a carrier of Cas9 RNP and evaluated genome editing activity in the neuron and the brain. A Cas9 RNP ternary complex with GUG-β-CDE (G3) was prepared by only mixing the components. The resulting complex exhibited higher genome editing activity than the complex with the dendrimer (G3), Lipofectamine 3000 or Lipofectamine CRISPRMAX in SH-SY5Y cells, a human neuroblastoma cell line. In addition, GUG-β-CDE (G3) enhanced the genome editing activity of Cas9 RNP in the whole mouse brain after a single intraventricular administration. Thus, GUG-β-CDE (G3) is a useful Cas9 RNP carrier that can induce genome editing in the neuron and brain.