Analysis of the Role of ABCG2 in Kidney Cell Activation by Monosodium Urate (MSU) Crystals through Transfection of a Specific sgRNA Using the CRISPR-Cas9 Technique

Abstract

Background/Objective

ABCG2's function as a urate transporter was deduced from genome-wide association studies and subsequent functional studies, which specifically demonstrated its importance in the elimination of uric acid in different tissues. Subsequent studies have shown associations between single nucleotide polymorphisms (SNPs) in ABCG2 and hyperuricemia and gout, particularly an SNP (rs2231142; Q141K), which has recently been linked to the disease. The Cas9-associated protein is an endonuclease that uses a guide sequence within a duplex RNA, tracrRNA:crRNA, also called single guide RNA (sgRNA). The technique that uses this enzyme can permanently and stably edit the genome, making it important to use it to silence the ABCG2 gene and examine how it affects inflammatory activation in gout models. Objective: Develop and analyze a specific sgRNA sequence to silence the gene expression of ABCG2 in HEK293T

Methods

The Horizon Discovery platform and its CRISPR Design Tool were used to obtain the sgRNA sequence in exon 8 of the ABCG2 gene. Transfection was performed by plating cells in 96-well plates until reaching 80% confluence, and then the culture medium was replaced with transfection medium containing the CRISPR-Cas9 system with the specific sequence and DharmaFECT as the transfection vehicle. Once transfection was completed, its efficacy was evaluated by assessing gene expression via qRT-PCR, and ABCG2 protein expression via western blot and immunofluorescence

Results

A decrease in both gene expression and protein expression (western blot) of ABCG2 has been found in some groups of transfected cells. Basal expression of ABCG2 was determined by immunofluorescence, and IL1beta expression with MSU at 24 hours was analyzed, as well as the expression of IL1beta by RT-PCR

Conclusion

Edited HEK293T cell lines have been obtained that express a lower amount of ABCG2 for subsequent sequencing