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Weiming Mao, Hannah Webber, Alvin Nguyen, Daniel Goan; Inhibition of TGFβ2 using CRISPR interference in the trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3487. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In primary open angle glaucoma (POAG) patients, glaucomatous intraocular pressure (IOP) elevation is due to pathological changes in the trabecular meshwork (TM). Our recent publication showed that there is histone hyperacetylation in the glaucomatous TM which may lead to elevated TGFβ2. However, gene specific histone modification has been technically challenging. Recently, the novel CRISPR interference technology has made this modification possible. The dCas9-KRAB system employs the mutant Cas9 enzyme (dCas9) which is able to bind specific DNA regions with the help of sgRNA but lacking DNA cleavage capability as well as the KRAB domain which is fused to dCas9 and is able to deacetylate histone proteins. We determined whether the CRISPRi/dCas9-KRAB system is useful in lowering TGFβ2 levels in the TM.
Three dCas9-KRAB expression systems were used: 1) a one-vector system containing the ubiquitin (UBC) promoter+dCas9-KRAB cassette and the U6 promoter-sgRNA cassette; 2) a two-vector system containing a vector with the EF1α promoter-dCas9-KRAB cassette and a vector with the U6 promoter-sgRNA cassette; 3) a two-vector system containing a vector with the SFFV promoter-dCas9-KRAB cassette and a vector with the U6 promoter-sgRNA cassette. sgRNA was designed using an online software targeting regions close to the transcriptional start site. Four sets of sgRNA were subcloned downstream of the U6 promoter. Vectors were transfected or co-transfected into the transformed GTM3 cell strains. Four days after transfection, cells were harvested for qPCR analysis and Western immunoblotting. The expression level of TGFβ was normalized to GAPDH using the delta-delta Ct method.
The expression of dCas9-KRAB and/or sgRNA did not show toxicity to GTM3 cells. qPCR analysis showed that the one vector system with one of the four sgRNAs has mild repression of TGFβ2 expression. In contrast, the 2 two-vector systems showed a dramatic repression of TGFβ2 in GTM3 cells.
The CRISPRi/dCas9-KRAB mediated inhibition of TGFβ2 expression is sgRNA as well as promoter dependent. Further studies are required to determine the specificity and suitability of this technology in other genes and primary human TM cells.
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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