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Naga pradeep Rayana, Weiming Mao; dCAS9-KRAB inhibits TGFβ2 in trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5140. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In primary open angle glaucoma (POAG), there is elevation in intraocular pressure due to the pathological changes in the trabecular meshwork (TM) cells. Our recent studies showed that histone hyper acetylation increases TGFβ2 in human TM (HTM) cells as well as perfusion cultured bovine eyes. Gene specific histone modification is technically challenging. This can be overcome by using CRISPR interference technology. The dCAS9-KRAB system employs the mutant CAS9 enzyme (dCAS9), which binds to specific DNA region with the help of sgRNA, but lacks DNA cleaving capacity, as well as the KRAB domain, which a histone deacetylase. In this study, we determined whether dCAS9-KRAB system is useful in lowering TGFβ2 expression in primary HTM cells and a transgenic TM cell line.
A two vector system is used which contains 1) A vector expressing dCAS9-KRAB with the EF1α or SFFV promoter; 2) A vector expressing sgRNA driven by the U6 promoter. sgRNA and dCAS9- KRAB were transfected into transformed GTM3 cells, and after four days cells were harvested for qPCR. The expression of TGFβ2 was normalized to GAPDH using the delta-delta Ct method. In primary HTM and some GTM3 cells, two lentiviral vectors which express sgRNA or dCAS9-KRAB were used for co-transduction. We also constructed a lentiviral vector which uses a CMV promoter to express a mutant human TGFβ2 fused with GFP, and used this vector to create a GTM3 line which stably expresses this fusion protein. These GTM3 cells were co-transfected with dCAS9-KRAB as well as sgRNA targeting the CMV promoter.
We first screened 24 sgRNAs targeting the promoter region of the TGFβ2 gene using GTM3 cells. Our screening showed that 4 sgRNAs could achieve a consistent inhibition of TGFβ2 by 30%- 50%. We then constructed lentiviral vectors for dCAS9 and the 4 sgRNAs, and compared their efficiency in primary HTM cells. qPCR showed that they decreased TGFβ2 by up to 46% compared to the non-targeting sgRNA as a negative control. We also screened 4 sgRNAs designed to target the CMV promoter of the transgenic GTM3 line. We observed up to 53% decrease in TGFβ2 expression.
dCAS9-KRAB inhibited TGFβ2 expression in both GTM3 and primary HTM cells. We will further determine the efficiency of this system in TM cells, perfusion cultured eyes, and in mouse eyes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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