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Luke A Wiley, Laura R Bohrer, Erin R Burnight, Elizabeth L Kennedy, Edwin M Stone, Mark A Greiner, Michael D Wagoner, Budd Tucker; Development of corneal endothelial cell-specific reporter constructs and a CRISPR-Cas9 genome editing approach to study Fuchs Dystrophy.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1444. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Fuchs dystrophy is the most common corneal endothelial cell disease and the leading indication for corneal transplantation. Fuchs is characterized by loss of corneal endothelial cells (CECs), thickening of Descemet’s membrane, corneal edema and impaired vision. Several genetic mutations have been shown to be associated with Fuchs including COL8A2 and TCF4. The aim of this study was to: 1) design promoter constructs suitable to monitor the differentiation of induced pluripotent stem cells (iPSCs) to CECs and 2) design CRISPR-Cas9 genome editing strategies to correct Fuchs-causing genetic mutations.
To generate iPSCs, fibroblasts from an individual with normal ocular history were virally transduced with OCT4, SOX2, KLF4 and c-MYC. We cloned lentiviral promoter constructs that were upstream of fluorescent reporter genes that are turned on at different stages of the iPSC to CEC differentiation: neural crest cells (NGFR/p75-E2-Crimson) and CECs (COL8A1-GFP and CHD2-tRFP). Constructs were transfected to HEK293 cells to test promoter activity. SgRNAs were designed to target the trinucleotide repeat expansion in TCF4 or mutations in COL8A2. The sgRNA oligos were cloned into bicistronic AAV vectors expressing a chimeric small guide and tracrRNA transcript and a human codon-optimized S. aureus Cas9. CRISPR-Cas9 constructs were transfected to HEK293 and tested for targeting efficiency using the T7E1 assay, TA cloning and Sanger sequencing.
Transfected promoter constructs were robustly expressed in HEK293 cells, including membrane localization of the reporter for CDH2. Efficient targeting specificity of the TCF4 and COL8A2 CRISPR-Cas9 constructs in HEK293 cells were shown using the T7E1 assay and cleaved PCR products, indicating CRISPR-Cas9-mutated DNA, were imaged on agarose gels. The cutting efficiency for an sgRNA targeting COL8A2 was 27%.
We have engineered promoter constructs that will be used in future experiments aimed at developing an efficient method to generate iPSC-derived CECs from control patients and those with Fuchs caused by genetic mutations in COL8A2 or TCF4. Use of CRISPR-Cas9-mediated genome editing to correct the mutations in Fuchs patient-specific iPSCs will yield corrected control lines for comparison to diseased iPSC-derived CECs.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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