Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair

Abstract

Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODNmediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy. Corneal dystrophy is a hereditary disease that causes corneal opacity; 22 types of corneal dystrophy are currently classified according to the International Committee for Classification of Corneal Dystrophies (IC3D)0 1. Among these, transforming growth factor-beta-induced (TGFBI) corneal dystrophies occur most frequently in East Asian populations 2. TGFBI corneal dystrophies are caused by a point mutation, or insertion or deletion of some frames, in TGFBI, which is located on chromosome 5q31 1,2. Various mutations in TGFBI cause corneal opacities with different phenotypes, such as granular corneal dystrophy (GCD), lattice corneal dystrophy (LCD), Reis-Bücklers corneal dystrophy (RBCD), and Thiel-Behnke corneal dystrophy (TBCD) 3. There are genotype-phenotype correlations in TGFBI corneal dystrophy; for example, the R124H mutation causes GCD type2 (GCD2), and the R124C mutation causes LCD type1 (LCD1) 2. Even though we know that the corneal opacities are caused by accumulated TGFBI protein (TGFBIp) 4 , the mechanism or pathophysiology is not yet clearly understood 5. Animal models help elucidate the pathophysiology of corneal dystrophies. Previously, Bustamante et al. used a lentiviral vector to generate knock-in mice overexpressing the human R555W mutation, which causes GCD type1 (GCD1) 6. However, R555W mutant mice did not show any corneal phenotype according to this report 6. Tgfbi-knockout mice also failed to show corneal abnormalities even after systemic depletion of Tgfbi expression 7. Recently, Yamazoe et al. established an R124H mutant transgenic mouse model showing corneal opacities 8. Ho...

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“…A granular corneal dystrophy type 2 mouse model was established via R124H mutation of Tgfbi and cross-sectional studies in this model revealed that genotype and age were associated with the incidence of corneal opacity [ 39 ]. The Tgfbi R124C mutant mouse model was established via CRISPR/Cas9 and is characterized by corneal opacity owing to the accumulation of mut-TGFBI and delayed epithelial wound healing similar to that observed in human patients with R124C LCD; though this model might not be a reliable as a LCD1 model due to the lack of amyloid deposition [ 40 ]. Currently, to the best of our knowledge, no animal models of human TBCD are available.…”

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

“…In a recent literature survey, Zhang (2021) found that at least a dozen animal models have been generated using CRISPR/Cas genome editing technology. These include mouse, rat, pig, and rabbit models for studying various human diseases, including human non‐small‐cell lung cancers (NSCLCs) ( Maddalo et al, 2014 ), hepatocellular carcinoma (HCC) ( Liu et al, 2017 ), Hutchinson-Gilford progeria syndrome (HGPS) ( Liu ZQ et al, 2018 ), corneal dystrophy ( Kitamoto et al, 2020 ), and X-linked dilated cardiomyopathy (XLCM) ( Liu ZQ et al, 2018 ). Fujihara et al (2020) successfully created a rat model by using CRISPR/Cas9 genome editing for studying complex behavioral changes during schizophrenia.…”

New Frontier in the Management of Corneal Dystrophies: Basics, Development, and Challenges in Corneal Gene Therapy and Gene Editing

“…73 Generating R124C mutation in TGFβl using CRISPR/Cas9-mediated homology-directed repair mechanism in zygotes of C57BL/6Ncr mice a novel transgenic mice model was generated mimicking human LCD. 74…”

“…Using Cas9/gRNA (px 458) plasmid and a single-stranded HDR donor template, R124H mutation was corrected with a higher gene correction efficiency of 20.6% in heterozygous cells and 41.3% in homozygous cells with no detectable off-target effects 73 . Generating R124C mutation in TGFβl using CRISPR/Cas9-mediated homology-directed repair mechanism in zygotes of C57BL/6Ncr mice a novel transgenic mice model was generated mimicking human LCD 74 …”