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Naga pradeep Rayana, Weiming Mao, Chenna Kesavulu Sugali, Jiannong Dai; Inhibition of TGFβ2-induced ocular hypertension using CRISPR interference. Invest. Ophthalmol. Vis. Sci. 2021;62(8):479.
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Studies have shown that elevated TGFβ2 induces pathological changes in the trabecular meshwork (TM) leading to ocular hypertension (OHT) which are similar to the phenotypes observed in primary open-angle glaucoma. Our published studies suggest that elevated TGFβ2 may be due to promoter region hyperacetylation in the TM. dCAS9 is a mutated form of CAS9, the key enzyme in the CRISPR system. When fused with KRAB, a repression domain, and guided by sgRNA, dCAS9-KRAB can repress gene expression without altering genomic DNA (CRISPR interference). In this study, we showed that CRISPR interference is a useful tool in decreasing intraocular pressure (IOP) by repressing TGFβ2 in mouse eyes.
Lentiviral dCAS9-KRAB and sgRNA expression vectors were co-transduced into TM cells at an MOI value of 1 to 50. On the fifth day post-transduction, cell lysate and condition medium were extracted and the expression of TGFβ2 and dCAS9 were determined using western immunoblotting. Lentiviral CRISPR interference vectors or control vectors were injected into mouse eyes followed by a second injection of adenoviral vectors expression active TGFβ2.
Our initial screening showed two guide RNAs from a pool of sgRNAs were able to decrease the expression of TGFβ2 compared to control sgRNA in GTM3 cells. We also found similar efficiency of the sgRNAs in the inhibition of TGFβ2 in primary TM cell strains. ChIP-qPCR showed enrichment of dCAS9-KRAB at the TGFβ2 promoter region. In addition to TM cells, we tested our system in a mouse model. We injected male (n=6) and female (n=6) mouse eyes with lentiviral vectors expressing dCAS9-KRAB together with sgRNA targeting the CMV promoter or non-targeting sgRNA as control. We then injected both eyes with Ad5-CMV-ΔhTGFβ2. We found that specific sgRNA expressing eyes did not develop OHT while control sgRNA expressing eyes developed OHT (p<0.05).
CRISPR interference can be used as an important tool in decreasing TGFβ2 and TGFβ2-induced OHT. It can be used as an alternative for siRNA.
This is a 2021 ARVO Annual Meeting abstract.
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