Using modern genome editing technology, specifically CRISPR-Cas9, to determine genes associated with the increased risk of rheumatic disease

Abstract

This project focussed on determining the functional mechanisms underlying non-coding DNA regions harbouring base-pair changes which increase the risk of developing psoriasis and psoriatic arthritis (PsA). The disease-associated variants in these non-coding regions, identified through GWAS analysis, are typically enriched within enhancers. These elements regulate genes over large genomic distances, and the variants within them are presumed to affect their function. To functionally dissect these regions, a variety of CRISPR-Cas9 technologies were utilised, including CRISPR activation, CRISPR interference and CRISPR-based deletions. This allowed both the determination of functionally relevant regions of these risk loci and identification of the genes which they regulate. The main focus of this PhD was a disease association at Chromosome 1p36.23, which had been implicated in the risk of both psoriasis and PsA. The disease-implicated DNA is ~200Kb away from the nearest coding gene. Previous data showed keratinocyte-specific chromatin interactions between the risk locus and a cluster of genes nearby, but it was not clear which of these genes was the true target. Characterisation of HaCat keratinocyte cells harbouring CRISPR-based deletions of putative functional elements within the risk locus, alongside CRISPRa and CRISPRi perturbations, showed that an enhancer element in this region – which contained a disease-associated variant – controlled expression of the gene ERRFI1: a negative regulator of epidermal growth factor receptor signalling. This gene has not previously been implicated in the pathogenesis of psoriasis or PsA. Linking ERRFI1 and the EGFR pathway to these diseases may therefore inform new potential treatment strategies in PsA and psoriasis. Investigation at 1p36.23 also provided additional mechanistic insight when activation of the enhancer locus with dCas9-VPR unexpectedly led to repression of the target gene and significant deposition of repressive H3K27Me3 histone marks both at the SNP locus and the enhancer locus. This may be to do with a cis-acting non-coding RNA at the locus being activated by the dCas9 system This thesis also details attempts to investigate a PsA risk association at the IL23R gene locus. This investigation provides a useful case study of the limitations of CRISPR-based investigations of GWAS risk loci.

Date of Award	31 Dec 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Stephen Eyre (Supervisor), Anne Barton (Supervisor), John Bowes (Supervisor) & Antony Adamson (Supervisor)