UMMS Affiliation

Program in Molecular Medicine; Division of Endocrinology, Metabolism and Diabetes, Department of Medicine; Graduate School of Biomedical Sciences

Publication Date

2018-11-02

Document Type

Article

Disciplines

Amino Acids, Peptides, and Proteins | Cell Biology | Cells | Cellular and Molecular Physiology | Genetic Phenomena | Nucleic Acids, Nucleotides, and Nucleosides | Nutritional and Metabolic Diseases | Therapeutics | Tissues

Abstract

RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a "browning" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed "CRISPR-delivery particles," denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease.

Keywords

CRISPR-delivery particle, CRISPR/Cas, adipocyte, browning, drug delivery system, fat tissue, gene deletion, guide RNA, metabolic disease, nanoparticle, ribonuclear protein (RNP), therapeutic strategy

Rights and Permissions

© 2018 Shen et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Publisher's PDF posted after 12 month as allowed by publisher's author rights policy at http://www.jbc.org/site/misc/edpolicy.xhtml#copyright.

DOI of Published Version

10.1074/jbc.RA118.004554

Source

J Biol Chem. 2018 Nov 2;293(44):17291-17305. doi: 10.1074/jbc.RA118.004554. Epub 2018 Sep 6. Link to article on publisher's site

Journal/Book/Conference Title

The Journal of biological chemistry

Related Resources

Link to Article in PubMed

PubMed ID

30190322

Available for download on Friday, September 06, 2019

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