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E. Maryam Maryam Hanser, Marina Zakharevich, Ricardo F Frausto, Austin Kassels, Yu Li, Anthony J Aldave; Generation of ZEB1-deficient human corneal endothelial cells using CRISPR-Cas9 as a model for posterior polymorphous corneal dystrophy 3. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5653.
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© ARVO (1962-2015); The Authors (2016-present)
To generate a human corneal endothelial cell (HCEnC) culture model of posterior polymorphous corneal dystrophy 3 (PPCD3), which is characterized by ZEB1 haploinsufficiency.
Editing of ZEB1 in an immortalized HCEnC line (HCEnC-21T) was accomplished using CRISPR-Cas9. To ensure effective knockout of all known ZEB1 alternative transcript variants, we targeted the Cas9 nuclease using a guide RNA (gRNA) to exon 4, which is shared by all known transcript variants. Optimal guide sequence and predicted off-target sites were obtained using the CRISPR Design web tool, which scores (0-100%) gRNA quality by accounting for off-target hits. HCEnC-21T were transfected with the pSpCas9(BB)-2A-Puro construct containing the Cas9 nuclease gene, gRNA and the puromycin selection marker. Subsequently, puromycin selection and limited dilution were performed to generate single cell clones. Clones were expanded and characterized for ZEB1 editing by Sanger sequencing and CRISP-ID, and for ZEB1 protein production by Western blotting (WB). Allele-specific sequencing was performed using PCR and TA cloning. Potential sites of off-target Cas9-mediated double-stranded DNA breaks were examined by Sanger sequencing.
Submission of exon 4 sequence to the CRISPR Design web tool resulted in 12 gRNA sequences. The highest scoring (72%) gRNA was predicted to potentially cleave at 182 off-target sites, 12 of which were in coding regions. Of the 77 clones generated and screened using CRISP-ID, 11 were selected for ZEB1 protein analysis. WB showed that 5 clones lacked the wild type ZEB1 protein and 3 clones expressed moderate ZEB1 protein levels compared with 3 clones that expressed higher ZEB1 levels. Three clones representing each of the three genotypes (ZEB1+/+, ZEB1+/- and ZEB1-/-) were selected for allele-specific sequencing and off-target screening. Allele-specific sequencing demonstrated that each clone possessed allele sequences consistent with the results obtained by WB. None of the three clones demonstrated indels at any of the top 9 off-target sites.
We demonstrate the utility of the CRISPR-Cas9 gene-editing tool to generate HCEnC deficient in ZEB1 as a model for PPCD3.
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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