Biology subjects

Adamson, A.

Publications and source records attributed to Adamson, A..

23 records · Page 2Linked to original sources

bioRxiv2019

Complex deletion and proximal reinsertion of a 150bp regulatory sequence in the mouse Csf1r promoter mediated by CRISPR-Cas9.

This paper describes a deletion/reinsertion event encountered in a genome-editing project using CRISPR-Cas9. The objective was to delete a 150bp enhancer region in the mouse Csf1r locus using a pair of guides and a homology-dependent repair (HDR) template. The editing was successful in generating a founder pup with the anticipated precise deletion. However, the deleted fragment and a duplicated copy of part of the HDR template was reinserted around 50bp downstream. The reinsertion event was recognised because the PCR primer site used in genotyping was duplicated, so that there were three PCR products in a heterozygous animal and two in a homozygote. The event we describe is more subtle and more difficult to detect than large-scale rearrangements reported by others. We suggest that any genomic deletion mediated by CRISPR-Cas9 needs to be confirmed by assessing the copy number in the genome.

molecular biology↗

bioRxiv2019

Simultaneous analysis of open chromatin, promoter interactions and gene expression in stimulated T cells implicates causal genes for rheumatoid arthritis

Genome-wide association studies have identified genetic variation contributing to complex disease risk. However, assigning causal genes and mechanisms has been more challenging because disease-associated variants are often found in distal regulatory regions with cell-type specific behaviours. Here, we collect ATAC-seq, Hi-C, Capture Hi-C and nuclear RNA-seq data in stimulated CD4+ T-cells over 24 hours, to identify functional enhancers regulating gene expression. We characterise changes in DNA interaction and activity dynamics that correlate with changes gene expression, and find that the strongest correlations are observed within 200 kb of promoters. Using rheumatoid arthritis as an example of T-cell mediated disease, we demonstrate interactions of expression quantitative trait loci with target genes, and confirm assigned genes or show complex interactions for 20% of disease associated loci, including FOXO1, which we confirm using CRISPR/Cas9.

genomics↗

bioRxiv2019

Chromatin-based techniques map DNA interaction landscapes in psoriasis susceptibility loci and highlight KLF4 as a target gene in 9q31

Genome-wide association studies (GWAS) have uncovered many genetic risk loci for psoriasis, yet many remain uncharacterised in terms of the causal gene and their biological mechanism in disease. Here, we use a disease-focused Capture Hi-C experiment to link psoriasis-associated variants with their target genes in psoriasis-relevant cell lines (HaCaT keratinocytes and My-La CD8+ T cells). We confirm previously assigned genes, suggest novel candidates and provide evidence for complexity at psoriasis GWAS loci. In the 9q31 risk locus we combine further epigenomic evidence to demonstrate how the psoriasis association forms a functional interaction with the distant (>500 kb) KLF4 gene. We use CRISPR activation coupled with RNA-seq to demonstrate how activation of psoriasis-associated enhancers upregulates KLF4 in HaCaT cells. Our study design provides a robust pipeline for following up on GWAS disease-associated variants, paving the way for functional translation of genetic findings into clinical benefit.

genomics↗

bioRxiv2018

Collagen assembly and turnover imaged with a CRISPR-Cas9 engineered Dendra2 tag

Electron microscopy has been the \"gold standard\" for studying collagen networks but dynamic information on how cells synthesise the networks has been lacking. Live imaging methods have been unable to distinguish newly-synthesised fibrils from pre-existing fibrils and intracellular collagen. Here, we tagged endogenous collagen-I using CRISPR-Cas9 with photoswitchable Dendra2 and demonstrate live cells synthesising, migrating on, and interacting with, collagen fibrils. This strategy is applicable for other long half-life proteins.

cell biology↗

bioRxiv2018

Circadian Clock Regulation of the Secretory Pathway

Collagen is the most abundant secreted protein in vertebrates that persists throughout life without renewal. The unchanging nature of collagen contrasts with observed continued collagen synthesis throughout adulthood and with conventional transcriptional and translational homeostatic mechanisms that replace damaged proteins with new copies. Here we show circadian clock regulation of procollagen transport from ER-to-Golgi and Golgi-to-plasma membrane by sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis, assembly and degradation affects only a pool of newly-synthesized collagen whilst maintaining the persistent collagen network. Disabling the circadian clock causes collagen accumulation and abnormal fibrils in vivo. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis in which a sacrificial pool of collagen is synthesized and removed to maintain tissue function.

cell biology↗