Generation of TGFBI knockout ABCG2+/ ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing

Eung Kweon Kim, Seunghyuk Kim, Yong Sun Maeng

Research output: Contribution to journalArticle

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 subclones, 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.

Original languageEnglish
Article numbere0211864
JournalPloS one
Volume14
Issue number2
DOIs
Publication statusPublished - 2019 Feb

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Stem cells
Guide RNA
stem cells
epithelial cells
Stem Cells
Clone Cells
Genes
Epithelial Cells
clones
genome
RNA
mutation
Mutation
exons
Exons
frameshift mutation
Gene therapy
transforming growth factors
Frameshift Mutation

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

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title = "Generation of TGFBI knockout ABCG2+/ ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing",
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 subclones, 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.",
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Generation of TGFBI knockout ABCG2+/ ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing. / Kim, Eung Kweon; Kim, Seunghyuk; Maeng, Yong Sun.

In: PloS one, Vol. 14, No. 2, e0211864, 02.2019.

Research output: Contribution to journalArticle

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