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

Jeanette M Metzger; Yuyuan Wang; Samuel S Neuman; Kathy J Snow; Stephen A Murray; Cathleen M Lutz; Viktoriya Bondarenko; Jesi Felton; Kirstan Gimse; Ruosen Xie; Dongdong Li; Yi Zhao; Matthew T Flowers; Heather A Simmons; Subhojit Roy; Krishanu Saha; Jon E Levine; Marina E Emborg; Shaoqin Gong

doi:10.1016/j.biomaterials.2022.121959

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

Biomaterials. 2023 Feb:293:121959. doi: 10.1016/j.biomaterials.2022.121959. Epub 2022 Dec 13.

Authors

Jeanette M Metzger¹, Yuyuan Wang², Samuel S Neuman¹, Kathy J Snow³, Stephen A Murray³, Cathleen M Lutz³, Viktoriya Bondarenko¹, Jesi Felton¹, Kirstan Gimse⁴, Ruosen Xie², Dongdong Li², Yi Zhao², Matthew T Flowers¹, Heather A Simmons¹, Subhojit Roy⁵, Krishanu Saha⁴, Jon E Levine⁶, Marina E Emborg⁷, Shaoqin Gong⁸

Affiliations

¹ Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, 53715, USA.
² Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, 53715, USA; Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA.
³ The Jackson Laboratory, Bar Harbor, ME, 04609, USA.
⁴ Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA.
⁵ Departments of Pathology and Neuroscience, University of California-San Diego, San Diego, CA, 92093, USA.
⁶ Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, 53715, USA; Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, 53715, USA.
⁷ Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, 53715, USA; Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, 53715, USA. Electronic address: emborg@wisc.edu.
⁸ Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, 53715, USA; Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA. Electronic address: shaoqingong@wisc.edu.

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 healthy 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.

Keywords: Brain; CRISPR; Genome editing; Nanocapsule; Nanoparticle; Neuron.

MeSH terms

Animals
Brain / metabolism
CRISPR-Cas Systems / genetics
Gene Editing* / methods
Mice
Nanocapsules*
Neurons / metabolism
Ribonucleoproteins / genetics
Ribonucleoproteins / metabolism

Substances

Nanocapsules
Ribonucleoproteins

Abstract

MeSH terms

Substances

Grants and funding