Dataset Information

Digenome for off-target analysis: Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

ABSTRACT: A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs’ endothelial corneal dystrophy (FECD), which can cause blindness through progressive loss of corneal endothelial cells. We established a novel procedure for achieving structural and functional rescue of the post-mitotic corneal endothelium without surgery, using CRISPR/Cas9-based postnatal gene editing in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) at a titer below the cytotoxic threshold, efficiently knocked down COL8A2 protein expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. Here, we evaluated the off-target activity of humanized gRNA targeting COL8A2 gene by a modified digenome analysis (Nat Methods 12, 237-243). Briefly, in vitro SpCas9 digested genomic DNA with/without gRNA are compared by deep sequencing. The target sequence is CGTCCACGGACGCCATGCT. In this study, we identified 13 candidate sites with homology to the gRNA and 8 candidate sites without homology to the gRNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE146997 | GEO | 2020/09/01

REPOSITORIES: GEO

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Project description:Transparency of the human cornea is necessary for vision. Fuchs Endothelial Corneal Dystrophy (FECD) is a bilateral, heritable degeneration of the corneal endothelium, and a leading indication for corneal transplantation in developed countries. While the early onset, and rarer, form of FECD has been linked to COL8A2 mutations, the more common, late onset form of FECD has genetic mutations linked to only a minority of cases. Epigenetic modifications that occur in FECD are unkonwn. Here, we report on and compare the DNA methyhlation landscape of normal human corneal endothelial (CE) tissue and CE from FECD patients using the Illumina Infinium HumanMethylation450 (HM450) DNA methylation array. We show that DNA methylation profiles are distinct between control and FECD samples. Differentially methylated probes (10,961) were identified in the FECD samples compared with the control samples, with the majority of probes being hypermethylated in the FECD samples. Genes containing differentially methylated sites were disproportionately annotated to ontological categories involving cytoskeletal organization, ion transport, hematopoetic cell differentiation, and cellular metabolism. Our results suggest that altered DNA methylation patterns may contribute to loss of corneal transparency in FECD through a global accumulation of sporadic DNA methylation changes in genes critical to basic CE biological processes Methylation of DNA is a key epigenetic mark that occurs in aging tissues. Altered DNA methlyation patterns have been observed in several late onset, and progressive ocular diseases including macular degeneration, glaucoma, and cataracts. While DNA methylation changes also occur in the common, late onset corneal dystrophy, FECD, has not been previously studied. Fuchs Endothelial Corneal Dystrophy (FECD) is a bilateral, heritable degeneration of the corneal endothelium, and a leading indicatioin for corneal transplantation in developed countries. Our study examined and compared the genome-scale DNA methylation profiels of corneal endothelial tissue from normal control and FECD patients using the Illumina Infinium HumanMethylation450 (HM450) DNA methylation array. We show that DNA methylation profiles are distinct between control and FECD samples. Differentially methylated probes (10,961) were identified in the FECD samples compared with the control samples, with the majority of probes being hypermethylated in the FECD samples. Genes containing differentially methylated sites were disproportionately annotated to ontological categories involving cytoskeletal organization, ion transport, hematopoetic cell differentiation, and cellular metabolism. Our findings suggest that alterations in DNA methylation may contribute to FECD pathogenesis by modifying the expression of genes with critical biological roles in the corneal endothelium. Our study has important clinical implications as FECD is a leading indication for corneal transplantationin the geriatric population. The effective medical treatment of FECD is a major unmet clinical challenge. Our findings suggest altered DNA methylation as a novel candidate therapeutic target in FECD

Project description:Fuchs’ endothelial corneal dystrophy (FECD) is a progressive vision impairing disease caused by thickening of Descemet’s membrane and gradual degeneration and loss of corneal endothelial cells. The aim of this study was to identify differentially expressed genes between FECD-affected and unaffected corneal endothelium to gain insight into the pathophysiological mechanisms underlying this disease. Microarray gene expression analysis was performed on total RNA from FECD-affected and unaffected corneal endothelium-Descemet’s membrane (CE-DM) specimens using the Illumina HumanHT-12 v3.0 expression array. RNA from pools of FECD-affected (n=3 per pool) and individual unaffected (n=3) specimens was used for comparison. Altered expression of a sub-set of differentially expressed genes was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in independent specimens. Bioinformatics analysis was performed using InnateDB to reveal functional relationships among the differentially expressed genes and molecular pathways involved in the disease. A total of 16,513 genes were found expressed in the corneal endothelium of which 142 genes were differentially expressed between FECD-affected and unaffected endothelium (log2 fold-change ≥1.5, corrected p-value ≤0.05). Most of the genes were up-regulated (126) and a small proportion down-regulated (16) in affected corneal endothelium. Of the twelve genes prioritised for validation, differential expression of 10 genes, including those ranked 57th and 81st by significance validated by qRT-PCR (8 up-regulated and 2 downregulated, corrected p ≤0.05), one gene showed a trend for up-regulation in affected endothelium, consistent with the microarray analysis and another was up-regulated in an independent study indicating robustness of the differential expression dataset. Bioinformatic analysis revealed significant over-representation of differentially expressed genes in extracellular matrix reorganisation, cellular remodelling, immune response, and inflammation. Network analysis showed functional inter-relatedness of the majority of the dysregulated genes and revealed known direct functional relationships between 20 of the genes; many of these genes have roles in macrophage differentiation, phagocytosis and inflammation. This is the second report of microarray gene expression analysis in FECD. This study revealed a set of highly dysregulated genes in the corneal endothelium in FECD. More than a third of the dysregulated genes in the disease have been discovered for the first time and thus are novel. The dysregulated genes strongly suggest the presence of phagocytic cells, most likely immune cells, and inflammation in corneal endothelium in the disease. This study provides a molecular framework for delineating the mechanisms underlying these cellular processes in FECD.

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Digenome for off-target analysis: Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

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