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

doi:10.1038/s41598-017-16308-2

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.

Highlights

Granular corneal dystrophy (GCD) is a bilateral, progressive, genetic, and non-inflammatory disease characterised by multiple granular deposits in the corneal stroma
Using the IC3D classification[1], GCD has two subtypes, both of which are classified as Category 1, i.e., causal point mutations have been identified in the transforming growth factor-beta-induced (TGFBI) gene, located on chromosome 5q312
To develop an efficient strategy to repair the genetic mutation in GCD using CRISPR/Cas[9], we used human cultured corneal keratocytes derived from an R124H GCD type 2 (GCD2) patient as a model system

Summary

Introduction

Granular corneal dystrophy (GCD) is a bilateral, progressive, genetic, and non-inflammatory disease characterised by multiple granular deposits in the corneal stroma. Homozygous patients may have an onset under 10 years old, and demonstrate a more rapid progression These progressive corneal opacities cause a loss of visual acuity. CRISPR/Cas[9] (clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein)-mediated genome editing has been increasingly applied to repair mutated genome sequences[10]. This versatile tool for genome engineering enables the induction of site-specific double-strand breaks (DSBs) using guide RNAs (gRNAs)[11,12,13,14,15]. The results of this study have important clinical implications given the lack of effective treatment options for GCD2

Methods

Results

Conclusion