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Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

ABSTRACT: Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

PROVIDER: PRJNA612701 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11309043_1.fastq.gz	Fastqsanger.gz
	SRR11309043_2.fastq.gz	Fastqsanger.gz
	SRR11309044_1.fastq.gz	Fastqsanger.gz
	SRR11309044_2.fastq.gz	Fastqsanger.gz
	SRR11309045_1.fastq.gz	Fastqsanger.gz

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Indel rate analysis:Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

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Indel rate analysis:Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

Project description: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, to determine the indel rate in mouse corneal endothelium, we performed deep sequencing of PCR products (including the target site) amplified from genomic DNA of corneal endothelium. We found that the indel rate was 23.7 ± 4.5% in mouse corneal endothelium. Most insertions were 1bp insertions (19.8 ± 4.0% in total reads), while 2bp deletions were the most frequent (1.0 ± 0.3% in total reads). We moreover found that A or T insertion was predominant, with the proportion of A:T:G:C being 48.7 : 44.6 : 1.8 : 4.9.

2020-09-01 | GSE146998 | GEO

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

Project description: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.

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

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

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Proteomics of Fuchs Endothelial Corneal Dystrophy support that the extracellular matrix of Descemet¹s membrane is Disordered

Project description:Fuchs’ endothelial corneal dystrophy is major corneal disorder in the western world affecting the innermost part of the cornea, which leads to visual impairment. The morphological changes observed in Fuchs’ endothelial corneal dystrophy is well described, however, much less in known of the pathology at the molecular level. As the morphological changes observed in the cornea is profound in the extracellular matrix we sought to determine in protein profiles and changes herein in the Descement’s membrane and endothelium layer of Fuchs’ endothelial conrneal dystrophy patients when compared to healthy control tissue. Using the extracted ion chromatogram label-free MS based quantification method we quantified approximately the 50 most abundant proteins of the Descemet’s membrane and endothelial layer in in patient and control tissue. In addition, using the isobaric tag for relative and absolute quantification MS method resulted in a total of 22 regulated proteins of which the majority were extracellular proteins known to be involved in proper assembly and modulation of the basement membrane in other tissues. Many of the regulated proteins were furthermore among the most abundant proteins quantified. The two MS methods performed here suggest altered arrangement of the extracellular matrix in Fuchs’ endothelial corneal dystrophy and provide new candidate proteins that may be involved in molecular mechanism of this disease.

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Gene expression in Fuchs endothelial corneal dystrophy and healthy cultured cells

Project description:Fuchs endothelial corneal dystrophy (FECD) is a vision impairing pathology affecting the endothelial cells of the cornea. To better understand the disease, we developed a method to cultivate FECD cells isolated from surgical specimens. Using gene profiling, we compared the mRNA profiles of passage 2 FECD cells with passage 2 non-pathological corneal endothelial cells isolated from eye bank donor corneas.

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