Application of the CRISPR interference method in regulating TGFβ2 in the trabecular meshwork

Introduction: Glaucoma is an eye disease that damages the optic nerve and leads to gradual loss of vision. Glaucoma is the second leading cause of blindness globally. The trabecular meshwork (TM) is an ocular tissue responsible for controlling and drainage the aqueous humor which is a fluid that fills the eye. In primary open angle glaucoma (POAG), the most frequent type of glaucoma, there is a dysfunction within the TM that decreases the outflow of aqueous humor and elevates intraocular pressure (IOP). Transforming growth factor beta 2 (TGFβ2) is a protein that controls cell growth, differentiation, proliferation, and apoptosis. Many studies have shown that elevated TGFβ2 induces glaucoma phenotypes in the eye, including elevated IOP. Therefore, lowering TGFβ2 levels in the TM is a potential therapeutic strategy for treating glaucomatous changes in the TM as well as lowering IOP. Since our published study showed that elevated TGFβ2 is likely due to histone hyperacetylation, the purpose of this study was to determine whether the novel CRISPR interference technology, which is able to deacetylate histones in a gene-specific manner, is suitable for the manipulation of TGFβ2 levels in the TM. Methods: Four sets of oligos were designed close to the transcriptional start site of the TGFβ2 gene using the online CRISPR sgRNA design tool (http://crispr.mit.edu/) for the construction of sgRNAs. These oligos were sub-cloned into the target sgRNA expression vector (Addgene). The dCas9-KRAB expression vector was purchased from Addgene. The sgRNA expression vector and dCas9-KRAB vector were co-transfected in transformed human TM cells (GTM3). Four days after transfection, we isolated mRNA and protein for quantitative PCR (qPCR) and Western immunoblotting analyses. Results: The expression of dCas9-KRAB and/or sgRNA did not show toxicity to GTM3 cells. qPCR analysis showed that the 2 two-vector system dramatically repressed the level of TGFβ2 in GTM3 cells. Conclusions: The CRISPR/dCAS9 interference method is effective in lowering the level of TGFβ2 in the HTM. Further studies are required to determine the specificity and suitability of this technology in other genes and primary human TM cells.