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Sai Santosh Kumar Bhuvanagiri, Balamurali Ambati, Hironori Uehara, Susie Choi, Xiaohui Zhang, Brain Bentley; The Disruption of Collagen8A2 gene using CRISPR/CAS9 system to treat Fuch’s Dystrophy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5298.
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© ARVO (1962-2015); The Authors (2016-present)
Fuch’s dystrophy is a progressive disease where the corneal endothelial cells gradually die causing corneal edema. Human genomic studies demonstrated an association of a Col8A2 (Collagen 8A2) mutation with early-onset Fuch’s dystrophy. Using this information, our purpose was to develop a method to prevent early-onset Fuch's dystrophy using CRISPR/CAS9 system to create an indel (insertion-deletion) in the start codon of this Col8A2 gene specifically in the corneal endothelial cells.
Cas9 gRNAs were designed to break mice Col8A2 gene by creating an indel in the ATG start codon of Col8A2 gene through non-homologous end rejoining. The designed gRNAs along with CRISPR/CAS9 were inserted into plasmid px330 and transfected to NIH3T3 cells. Then the region including the exon3 of Col8A2 genomic DNA was amplified by PCR. To confirm the indel, PCR products were digested by CviAII restriction enzyme, which can only cut the intact start codon of Col8A2 gene, and were run on the agarose gel. Adenovirus encoding Cas9 and the same gRNA was created and injected (different quantities) into the anterior chamber of mice corneas.The indel at the start codon was checked genomically from the endothelial layer and epithelial cell layer.
The PCR product from gDNA of px330-gRNA transfected NIH3T3 showed the band that indicated the indel of ATG start codon of Col8A2 gene. This was confirmed by DNA sequencing. The in vivo genomic analysis showed that around 50% of the Col8A2 start codon was altered after adenovirus injection in the mice corneal endothelial cells with ideal concentration of 6.25x106 gc (genomic content) of adenovirus vector. The start codon of Col8A2 gene was unaltered in the mice corneal epithelial cells and stroma.
From previous studies, it is known that COL8A2 gene is indicated in the cause of early-onset Fuch’s dystrophy. From our research, it is found that CRISPR/CAS9 and adenovirus can be effectively used to alter the start codon of Col8A2 gene specifically in the target corneal endothelial cell and possibly prevent early-onset Fuch’s dystrophy. Our research will further look at the altered Col8A2 gene expression due to the altered start codon of Col8A2 in the cornea and the morphological differences over time in the mice cornea.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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