CRISPR/Cas9-based liver-derived reporter cells for screening of mPGES-1 inhibitors

Zhanfei Chen,Xiaoling Cai,Man Li,Linlin Yan,Luxi Wu,Xiaoqian Wang,Nanhong Tang

doi:10.1080/14756366.2019.1587416

Zhanfei Chen, Xiaoling Cai + Show 5 more

Open Access

https://doi.org/10.1080/14756366.2019.1587416

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Abstract

mPGES-1 is a terminal rate-limiting enzyme responsible for inflammation-induced PGE2 production. The inhibition of mPGES-1 has been considered as a safe and effective target for the treatment of inflammation and cancer. However, a specific, efficient, and simple method for high-throughput screening of mPGES-1 inhibitors is still lacking. In this study, we developed a fluorescence imaging strategy to monitor the expression of mPGES-1 via CRISPR/Cas9 knock-in system. Immunofluorescence colocalisation, Sanger sequencing, RNAi, and IL-1β treatment all confirmed the successful construction of mPGES-1 reporter cells. The fluorescence signal intensity of the reporter cells treated with four conventional mPGES-1 inhibitors was considerably attenuated via flow cytometry and fluorescent microplate reader, demonstrating that the reporter cells can be used as an efficient and convenient means for screening and optimising mPGES-1 inhibitors. Moreover, it provides a new technical support for the development of targeted small molecule compounds for anti-inflammatory and tumour therapy.

Highlights

Prostaglandin E (PGE2), a mediator of the body’s inflammatory response, is closely related to many inflammation-related diseases, such as cardiovascular, joint, and neurodegenerative diseases, as well as cancer[1,2,3]
To construct mPGES-1 reporter cells, we applied the principle of CRISPR/Cas[9] knock-in gene editing (Figure 1(A)) to cotransfect mPGES-1 sgRNA recombinant vector with a homologous recombinant donor vector in liver-derived cells
Based on the principle of the CRISPR/Cas[9] system knock-in and the HDR pathway, we inserted the tdTomato gene into the stop codon position of the chromosomal mPGES-1 gene. The expression of both genes was under the transcriptional regulation of the mPGES-1 promoter

Summary

Introduction

Prostaglandin E (PGE2), a mediator of the body’s inflammatory response, is closely related to many inflammation-related diseases, such as cardiovascular, joint, and neurodegenerative diseases, as well as cancer[1,2,3]. In non-inflammatory response, the body relies on COX-1 (non-inducible), cPGES, and mPGES-2 to produce basal levels of PGE2 to maintain organs’ physiological function. AA produces an excess of prostaglandin H2 (PGH2) via COX-2 enzyme reaction. The use of COX-2 inhibitors can reduce the production of PGE2 and achieve anti-inflammatory effects. Studies have shown that COX-2-specific inhibitors have many side effects (causing cardiovascular disease or gastrointestinal bleeding)[8,9], which showcase the urgent need for the discovery of novel potent and safe anti-inflammatory drugs

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