Background/Purpose: The majority of trait-associated SNPs occur in noncoding regions and are enriched in enhancers. GWAS have identified numerous genetic variants associated with Systemic Lupus Erythematosus (SLE), but mechanistic insights remain limited, particular for noncoding polymorphisms. miR-146a is a negative regulator of the interferon pathway. In SLE patients, the expression of miR-146a is low and contributes to the abnormal activation of interferon pathway. rs2431697 is associated with the miR-146a expression level and located approximately 15.3 kb upstream of miR-146a exon 1, but how this risk variant regulates miR-146a expression and functionally contributes to the underlying pathogenesis is still unclear. Now, the application of CRISPR-Cas9 for genome engineering provides a new approach to study the function of disease susceptibility variants. This study was undertaken to investigate the role of rs2431697-containing region in the regulation of miR-146a expression by CRISPR-Cas9 technology.

Methods: To confirm whether rs2431697-containing region is a distal enhancer, we carried out ATAC-seq and formaldehyde-assisted isolation of regulatory elements (FAIRE) to test chromatin accessibility of this region. Chromatin immunoprecipitation (ChIP) followed by qRT–PCR (ChIP-qRT–PCR) was adopted to probe the enrichment of active enhancer makers-H3K4me1 and H3K27ac. SAM system is a dCas9-based transcription activation system comprising of dCas9-VP64 and MS2–p65–HSF1 complex, which can transactivate target genes from distal enhancers. We also take this system to test the function of this region. To detect whether rs2431697-containing region is actually involved in the regulation of miR-146a expression, CRISPR-Cas9 technology was used to generate a 30 bp-deletion of the genomic region containing rs2431697 in U937 cells and miR-146a expression was analyzed by TaqMan microRNA assay. We also generated rs2431697 T allele and rs2431697 C allele containing cell clone in U937 cells by CRISPR-Cas9 induced homology-directed repair, and using bioinformatics to predict the SNP-specific binding transcription factor.

Results: ChIP-qRT–PCR analysis showed that rs2431697-containing region was enriched with H3K27ac and H3K4me1 modification. ATAC-Seq and FAIRE-qRT-PCR indicated that the region was open. Additionally, dCas9-vp64-SAM system could activate miR-146a expression based on the guide RNAs around this region. Importantly, the generation of 30 bp-deletions comprising rs2431697 dramatically reduced the miR-146a expression at transcriptional level of up to 10-fold, but had no effect on the expression of the neighbor gene PTTG1. rs2431697 T allele and C allele comprising cell clone had different miR-146a expression level at both native and TNFα stimulation. Bioinformatics analysis suggested that rs2431697 T and C allele may have differential RelA binding.

Conclusion: Using CRISPR-Cas9 technology, we first indicate that rs2431697-containing region participates in the regulation of miR-146a expression. We also demonstrate that rs2431697 is located in a distal enhancer and has differential transcription factor binding that modulates miR-146a expression involving in the pathogenesis of SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-a-functional-susceptible-variant-in-distal-enhancer-of-mir-146a-by-crispr-cas9/