Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair

Taketani Yukako, Kitamoto Kohdai, Sakisaka Toshihiro, Kimakura Mikiko, Toyono Tetsuya, Yamagami Satoru, Amano Shiro, Kuroda Masahiko, Tara Moore, Usui Tomohiko, Ouchi Yasuo

Research output: Contribution to journalArticle

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Abstract

Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases.
LanguageEnglish
Article number16713
JournalScientific Reports
Volume7
Issue number1
Early online date1 Dec 2017
DOIs
Publication statusE-pub ahead of print - 1 Dec 2017

Fingerprint

Corneal Keratocytes
Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Mutation
Corneal Diseases
Genes
Corneal Stroma
Inborn Genetic Diseases
Heredity
Oligodeoxyribonucleotides
Point Mutation
Transforming Growth Factor beta
Plasmids
Chromosomes
Tissue Donors
Therapeutics

Keywords

  • CRISPR/Cas9
  • TGFBI
  • Gene

Cite this

Yukako, Taketani ; Kohdai, Kitamoto ; Toshihiro, Sakisaka ; Mikiko, Kimakura ; Tetsuya, Toyono ; Satoru, Yamagami ; Shiro, Amano ; Masahiko, Kuroda ; Moore, Tara ; Tomohiko, Usui ; Yasuo, Ouchi. / Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair. In: Scientific Reports. 2017 ; Vol. 7, No. 1.
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abstract = "Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6{\%} in heterozygous cells and 41.3{\%} in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases.",
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Yukako, T, Kohdai, K, Toshihiro, S, Mikiko, K, Tetsuya, T, Satoru, Y, Shiro, A, Masahiko, K, Moore, T, Tomohiko, U & Yasuo, O 2017, 'Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair', Scientific Reports, vol. 7, no. 1, 16713. https://doi.org/10.1038/s41598-017-16308-2

Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair. / Yukako, Taketani; Kohdai, Kitamoto; Toshihiro, Sakisaka; Mikiko, Kimakura; Tetsuya, Toyono; Satoru, Yamagami; Shiro, Amano; Masahiko, Kuroda; Moore, Tara; Tomohiko, Usui; Yasuo, Ouchi.

In: Scientific Reports, Vol. 7, No. 1, 16713, 01.12.2017.

Research output: Contribution to journalArticle

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AU - Yukako, Taketani

AU - Kohdai, Kitamoto

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AU - Mikiko, Kimakura

AU - Tetsuya, Toyono

AU - Satoru, Yamagami

AU - Shiro, Amano

AU - Masahiko, Kuroda

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AU - Tomohiko, Usui

AU - Yasuo, Ouchi

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N2 - Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases.

AB - Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases.

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KW - TGFBI

KW - Gene

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