CRISPR-Cas9 editing of TNFAIP3 variants in salivary gland epithelial cells to study Sjögren's disease pathogenesis

Abstract

Sjögren's disease (SD) is a systemic autoimmune disease that particularly affects the salivary and lacrimal glands, causing sicca symptoms. Genetic polymorphism in the TNFAIP3 gene has been implicated in the pathogenesis of SD. In this study, we aimed to functionally determine the impact of two specific single-nucleotide polymorphisms (SNPs) in TNFAIP3, rs6920220 (G/A) and rs2230926 (T/C/G), on the pathogenesis of SD. Using CRISPR-Cas9, we edited human salivary gland epithelial cells (SGECs) to incorporate TNFAIP3 SNPs rs6920220 (G/A) and rs2230926 (T/C/G) and co-cultured them with Jurkat cells. We performed assays to examine gene expression, inflammatory cytokine levels, and related signaling pathways to investigate the effects of these genetic variants on TNFAIP3 function and cellular response. Our results demonstrated that these SNPs reduced TNFAIP3 expression, increased NF-κB activation, and elevated pro-inflammatory cytokine production. These findings provide strong evidence for the functional significance of these genetic variants in the pathogenesis of SD and underscore the utility of CRISPR-Cas9 technology in elucidating genetic contributions to autoimmune disorders.

Keywords: CRISPR-Cas9; NF-κB; Sjögren’s disease; TNFAIP3; autoimmune diseases; salivary gland; single-nucleotide polymorphisms.

FIGURE 1

(A) TNFAIP3-associated risk factor SNP rs6920220 (G/A) and its reference sequence. (B) TNFAIP3-associated risk factor SNP rs2230926 (T/C/G) and its reference sequence.

FIGURE 2

(A) Relative fluorescence of WT and Mut probes targeting the rs6920220 (G/A) site within the forward and reverse primer-amplified region of genomic DNA from CRISPR-Cas9-edited SGECs. (B) Relative fluorescence of G, C, and T probes at the rs2230926 (T/C/G) site within the corresponding PCR-amplified region of genomic DNA from edited SGECs. Each sample was analyzed in two independent runs, with three technical replicates per condition (n = 3 wells per group).

FIGURE 3

(A) i–iv, Relative mRNA fold changes in TNFAIP3, NF-κB, TGF-β, pro-inflammatory cytokines (IL-1β, IL-6, and IL-8), and immune-related genes (PKR, IRF1, IRF5, CXCL10, OAS1, IL-4, IL-10, IL-12, and IL-13) in SGECs with and without CRISPR editing. (B) i–iv, Corresponding expression levels in Jurkat cells co-cultured with SGECs. Gene expression was normalized to the housekeeping gene GAPDH. Data represent means ± SD from two biological replicates, each with three technical replicates (n = 3). Statistical significance was determined using Student’s t-test.