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• W2912792401 abstract "Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor β-induced (TGFBI) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI-targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy." @default.
• W2912792401 created "2019-02-21" @default.
• W2912792401 creator A5002319303 @default.
• W2912792401 creator A5025083393 @default.
• W2912792401 creator A5072734871 @default.
• W2912792401 date "2019-02-12" @default.
• W2912792401 modified "2023-10-12" @default.
• W2912792401 title "Generation of TGFBI knockout ABCG2+/ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing" @default.
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• W2912792401 doi "https://doi.org/10.1371/journal.pone.0211864" @default.
• W2912792401 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6372159" @default.
• W2912792401 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30753226" @default.
• W2912792401 hasPublicationYear "2019" @default.
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