SCIENTIfIC REPORTS | 7: 16713 | DOI: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
, Kohdai Kitamoto
, Toshihiro Sakisaka
, Mikiko Kimakura
, Satoru Yamagami
, Shiro Amano
, Masahiko Kuroda
, Tara Moore
& Yasuo Ouchi
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 rst 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-specic
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 eciency was 20.6% in heterozygous cells and 41.3% in
homozygous cells. No o-target eects were detected. These results reveal a new therapeutic strategy
for GCD2; this method may also be applicable to other heredity corneal diseases.
Granular corneal dystrophy (GCD) is a bilateral, progressive, genetic, and non-inammatory disease character-
ised by multiple granular deposits in the corneal stroma. Using the IC3D classication
, GCD has two subtypes,
both of which are classied as Category 1, i.e., causal point mutations have been identied in the transforming
growth factor-beta-induced (TGFBI) gene, located on chromosome 5q31
. TGFBI, also called keratoepithelin or
Big-h3, is 68-kDa protein found in the extracellular matrix of human tissues. It is particularly abundant in the
ere are two clinical types of GCD, GCD1 and GCD2. Although originally described in a family from the
Italian region of Avellino, the R124H mutation associated with GCD2 is occurs in unrelated individuals in all
populations studied and is the most common type in Asia, including Japan
. In GCD2, discrete grey-white
granular deposits (hyaline) with snowake, star, or disk shapes are detected in the corneal stroma at an early
and amyloid deposits are observed in elder patients in deeper stroma
. GCD2 has a diuse anterior stromal
haze between the typical granular opacity. e haze may be caused by amyloid deposits, which are thought to
be similar to Gelatinous drop-like CD, instead of the linear opacity seen in the early stage of lattice dystrophy
Compared to heterozygous patients, homozygous patients may have an onset under 10 years old, and demon-
strate a more rapid progression. ese progressive corneal opacities cause a loss of visual acuity. To avoid visual
impairment in GCD2, phototherapeutic keratectomy (PTK) is a major treatment option. However, multiple
Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Itabashi Hospital, Tokyo, Japan.
Inoue Eye Hospital, Tokyo, Japan.
Department of Molecular Pathology, Tokyo
Medical University, Tokyo, Japan.
Biomedical Sciences Research Institute, Centre for Molecular Biosciences, Ulster
University, Coleraine, Northern Ireland.
Avellino Labs, Menlo Park, CA, USA.
Department of Mucosal Immunology,
School of Medicine, Chiba University, Chiba, Japan. Correspondence and requests for materials should be addressed
to T.U. (email: firstname.lastname@example.org) or Y.O. (email: email@example.com)
Received: 28 February 2017
Accepted: 6 November 2017
Published: xx xx xxxx