P073 Functional investigation of Osteoarthritis-associated non-coding variants using CRISPR/Cas9-mediated perturbation of active enhancer regions
Authors: Frantzeskos Antonios , Srinivasan Jhanavi , Morris Stanley , Shi Chenfu , McGovern Amanda , Richards Ellie , Rossi Stefano , Eyre Stephen , Orozco Gisela 
Published: April 2024

Abstract

Background/Aims
OA is a common, complex disease, affecting millions of people worldwide with no effective treatments. Recent studies have implicated over 100 regions of the genome that carry DNA changes that increase the risk of developing OA. Although these findings have advanced our understanding of the disease, implicating biological pathways and cell types as being important in disease, there is still much work required to understand the mechanism by which risk variants cause OA and fully translate the findings. This is primarily due to the nature of these DNA risk variants, often located outside gene coding regions, in regions that control gene activity. Here we use CRISPR technology to investigate two DNA regions implicated in OA risk to determine the target genes that are regulated and the likely cell types in which they operate.

Methods
We annotated OA associated SNPs utilising in-house ATAC-seq for open chromatin regions and CUT&Tag for H3K27ac marks generated for the first time here in two chondrocyte cell lines (TC28a2 and SW1353), along with H3K27ac marks from primary chondrocytes derived from the ENCODE database. Additionally we focused on regions which had genes expressed in chondrocytes within 1Mb of the lead SNP. Together this information guided prioritisation of OA regions and residing SNPs for functional validation. The two chondrocyte cell lines were engineered to constitutively express either CRISPR activation (dCas9-VPR) or CRISPR inhibition (dCas9-MeCP2-KRAB) machinery and subsequently designed guide RNAs targeted the prioritised SNPs. This enabled the activation or repression of regulatory regions, with the resultant effect on gene expression of potential target genes measured through qPCR.

Results
Through functional annotation of OA-associated regions, we focused on two SNPs prioritised across two distinct regions: rs1149611 in the TSKU region and rs3795159 in the UQCC1 region. These SNPs coincided with active chromatin marks in the chondrocyte cell lines and were targeted by CRISPR activation or inhibition systems in both chondrocyte cell lines. Both activation and inhibition of rs3795159 in the SW1353 chondrocyte cell line resulted in significant differential expression of the UQCC1 gene. Similarly, activation of rs1149611 upregulated TSKU in this cell line, whilst deletion of the SNP region downregulated TSKU expression in the Tc28a cell line. UQCC1 is a fibroblast growth factor, and TSKU is an inhibitor of TGFB1, and has been implicated in bone growth.

Conclusion
Although these results should be replicated in primary chondrocytes, here we determine firm evidence that DNA regions containing OA risk SNPs can regulate UQCC1 and TSKU in cell lines. This implicates these genes, and biological pathways, being perturbed prior to disease onset, and as such constitute novel targets for OA intervention.

Disclosure
A. Frantzeskos: None. J. Srinivasan: None. S. Morris: None. C. Shi: None. A. McGovern: None. E. Richards: None. S. Rossi: None. S. Eyre: None. G. Orozco: None.