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

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

PROVIDER: PRJNA612702 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11309045_1.fastq.gz	Fastqsanger.gz
	SRR11309045_2.fastq.gz	Fastqsanger.gz
	SRR11309046_1.fastq.gz	Fastqsanger.gz
	SRR11309046_2.fastq.gz	Fastqsanger.gz
	SRR11309047_1.fastq.gz	Fastqsanger.gz

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Similar Datasets

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

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

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

| PRJNA612701 | ENA

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 progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

| S-EPMC8216720 | biostudies-literature

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.

2020-09-01 | GSE146997 | GEO

Genetics of Fuchs Corneal Dystrophy

Project description:Genetics of Fuchs Corneal Dystrophy

| PRJNA545550 | ENA

Genetics of Fuchs Corneal Dystrophy

Project description:Genetics of Fuchs Corneal Dystrophy

| PRJNA545551 | ENA

Serial analysis of gene expression in the corneal endothelium of Fuchs' dystrophy

Project description:PURPOSE: To compare the gene expression profiles of normal human corneal endothelium with Fuchs' corneal endothelium, by using serial analysis of gene expression (SAGE). METHODS: Three pairs of normal human corneas were obtained from eye banks. Thirteen bisected Fuchs' corneal buttons were processed at the time of corneal transplantation. The endothelia of normal and Fuchs'-affected corneas were stripped, and total RNA was isolated. Serial analysis of gene expression (SAGE) was performed to identify and quantify gene transcripts. Genes over- and underexpressed by Fuchs' endothelium were limited to P < 0.01 by the method of Audic and Claverie. RESULTS: A total of 19,136 tags were identified with 9,530 from normal and 9,606 from Fuchs' endothelium. The expression of 18 transcripts was upregulated, and 36 transcripts were downregulated in Fuchs' endothelium compared with normal tissue. Upregulated transcripts included serum amyloid A1 and A2, metallothionein, and apolipoprotein D. Of the downregulated transcripts, 26 matched known genes, 3 matched expressed sequence tags (ESTs), and 7 were unknown to current databases. One downregulated transcript involved a newly reported bicarbonate transporter. Decreased transcripts related to antioxidants and proteins conferring protection against toxic stress were noted in Fuchs' versus normal endothelium including nuclear ferritin, glutathione S-transferase-pi, and heat shock 70-kDa protein. Nine different SAGE tags matching mitochondrial sequences accounted for 25% of the ESTs that were decreased in Fuchs' endothelium. CONCLUSIONS: SAGE analysis comparing normal to Fuchs' endothelium demonstrates diminished expression of mitochondrial, pump function, and antiapoptotic cell defense genes. A complete report of gene expression profiles of normal human corneal endothelium can be found in PMID 12583793. Keywords: other

2003-07-16 | GSE505 | GEO

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

Project description:This SuperSeries is composed of the SubSeries listed below.

2020-09-01 | GSE146999 | GEO

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

| PRJNA612703 | ENA

Transcriptome analyses of human corneal endothelial cell lines derived from patients with Fuchs Endothelial Corneal Dystrophy

Project description:We report differential gene expression profiles of the corneal endothelial cells from Fuchs dystrophy patients with or without the TCF4 gene mutation

2025-12-09 | GSE285203 | GEO

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