Mimicking TGFBI Hot-Spot Mutation Did Not Result in Any Deposit Formation in the Zebrafish Cornea

Abstract

Purpose

Mutations in transforming growth factor beta-induced (TGFBI) protein are associated with a group of corneal dystrophies (CDs), classified as TGFBI-associated CDs, characterized by deposits in the cornea. Mouse models were not proper in several aspects for modelling human disease. The goal of this study was to generate zebrafish mutants to investigate the corneal phenotype and to decide whether zebrafish could be a potential model for TGFBI-associated CDs.

Methods

The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3 months and 1 year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits.

Results

We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L + del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3 months and 1 year of age.

Conclusion

This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1 year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.

Keywords:

• CRISPR/Cas9
• TGFBI
• zebrafish
• corneal dystrophy
• genome editing

Acknowledgments

We would like to thank Dr. Adife Gülhan Ercan Sencicek, Masonic Medical Research Institute, USA for sharing experience during zebrafish genome editing experiments and Dr. Gaurav K Varshney, Genes & Human Disease Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA for providing Cas9 VQR variant plasmid. New paragraph: use this style when you need to begin a new paragraph.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author, [FYT], upon request.

Additional information

Funding

This work was supported by Scientific Research Projects (BAP) Coordination Unit of Hacettepe University (Grant number: TAY-201712735).