CRISPR-delivery particles targeting nuclear receptor–interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure

Yuefei Shen,Felipe Henriques,Jason K Kim,Mark Kelly,Randall H Friedline,Emmanouela Tsagkaraki,Jessica L Cohen,Michael P Czech,Sarah M Nicoloro,Batuhan Yenilmez,Xiaodi Hu,Yvonne J.K Edwards

doi:10.1074/jbc.ra118.004554

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.

Highlights

These studies were supported by National Institutes of Health Grants DK103047 and DK030898 and a grant from the International Research Alliance of the Novo Nordisk Foundation Center for Metabolic Research
Unlike White adipose tissue (WAT), brown adipose tissue (BAT) is composed of brown adipocytes that display a high capacity for fat oxidation and a high number of mitochondria containing uncoupling protein 1 (UCP1) for nonshivering thermogenesis that plays a beneficial role in metabolism [38]
We report the preparation of CRISPR-delivery particles (CriPs) that can deliver CRISPR-associated protein 9 (Cas9) protein bound to single-guide RNA (sgRNA) to mediate gene deletion in vitro and in vivo (Fig. 1)

Summary

Introduction

These studies were supported by National Institutes of Health Grants DK103047 and DK030898 and a grant from the International Research Alliance of the Novo Nordisk Foundation Center for Metabolic Research Cas9 –sgRNA RNP delivery could circumvent the safety problems associated with plasmid delivery, such as uncontrolled integration of DNA segments into the host genome and unwanted immune response to plasmids encoding Cas protein and sgRNA [6]. Systemic administration of Cas mRNA and sgRNA loaded into a lipid nanoparticle has been reported to achieve robust and persistent genome editing in vivo [24], but Cas9 –sgRNA RNPs have not been used systemically in vivo using a fully nonviral delivery system. We developed a novel CRISPR delivery system, denoted CRISPR-delivery particles (CriPs), composed of nano-size complexes of the CRISPR components Cas protein and sgRNA targeting a gene of interest, complexed with an EndoPorter (EP) peptide through electrostatic complexation.

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Conclusion