Background/Purpose: Findings from genome wide association studies in complex diseases indicate over 90% of genetic variants associated with risk of developing disease are found outside protein coding regions, suggesting regulation of gene expression is key to disease susceptibility. For rheumatoid arthritis (RA) it has been demonstrated that risk variants are found in gene regulation regions, and are significantly enriched in T-cell specific enhancers. In addition, a significant proportion of associated variants lay some distance from the nearest gene and enhancers may not necessary regulate the closest gene, effectively ‘skipping’ genes. Using chromatin conformation technology (HiC) we have demonstrated that an enhancer region intronic of the COG6 gene, containing variants associated with RA, make robust physical contact with the promoter of FOXO1, almost 1Mb away on the linear chromosome. COG6 is not an obvious candidate risk gene for RA, whilst FOXO1 is involved in T-cell development and shown to be over expressed in RA synovium. The challenge now is to provide empirical evidence that the enhancer found within COG6 does regulate FOXO1 expression, and how an RA risk genetic background affects this regulation

Use CRISPR-Cas9, to perturb the COG6 intronic enhancer region and measure the downstream effect on the expression of FOXO1.

Methods:

We utilised a modified form of the Cas9 enzyme, dead Cas9 (dCas9), that can precisely target DNA, but will not induce a cut. Using the dCas9 attached to either enhancers (p300) or repressors (KRAB) of expression we investigated how perturbation of the enhancer intronic of COG6 changed the expression of FOXO1.

We designed 3 guides across the COG6 enhancer, and transduced a cell line (HEK293) using a lentiviral dCas9 CRISPR system, with either dCas9-KRAB or dCas9-p300 and each of the three guides. We cultured the cells until 70-80% confluent, GFP sorted the cells and then extracted RNA. A quantitative PCR was performed (QuantStudio) for both COG6 and FOXO1 gene transcript expression and normalised to housekeeping genes.

Results: Up to 90% of HEK cells were tranduced with the dCas9 enzyme and guide, and these were sorted by FACS using GFP to sort the top 60% . The 3 guides gave consistently increased levels of FOXO1 expression with the dCas9-p300, compared to both control and dCas9-KRAB (p=0.02). This was particular evident for guide 3, with a 40% increase (p300) and 10% decrease (KRAB) of FOXO1 expression observed. Expression of COG6 was also perturbed, but in a less consistent manner, with both increase and decrease expression for KRAB and p300

Conclusion:

Over 90% of HEK cells were tranduced with the dCas9 enzyme and guide, and these were sorted by FACS using GFP to sort the top 60% . The 3 guides gave consistently increased levels of FOXO1 expression with the dCas9-p300, compared to both control and dCas9-KRAB (p=0.02). This was particular evident for guide 3, with a 40% increase (p300) and 10% decrease (KRAB) of FOXO1 expression observed. Expression of COG6 was also perturbed, but in a less consistent manner, with both increase and decrease expression for KRAB and p300.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/transcriptional-perturbation-of-ra-risk-enhancer-by-crispr-deadcas9-regulates-long-range-gene-targets/