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

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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.
Original languageEnglish
Article number16713
Pages (from-to)1-7
Number of pages7
JournalScientific Reports
Volume7
Issue number1
DOIs
Publication statusPublished - 1 Dec 2017

Keywords

  • CRISPR/Cas9
  • TGFBI
  • Gene

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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, 7(1), 1-7. [16713]. https://doi.org/10.1038/s41598-017-16308-2