Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins

ABSTRACT Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo . SIGNIFICANCE STATEMENT Modifying the DNA of cells in the brain could present opportunities for new treatments of neurological diseases. In this report, we describe a nanocapsule system designed to deliver the elements needed to modify the DNA of brain cells, also known as genome editing. These nanocapsules are created by chemically encapsulating the genome editing components, such that the nanocapsules are stable when prepared and biodegradable to release their payload upon entering cells. When injected into the mouse brain, our research shows that the nanocapsules lead to safe and efficient editing of DNA in neurons.