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Zhengping Hu, Na Chen, Patricia A D'Amore, Hetian Lei; Inactive Cas9 blocks vitreous-induced contraction of retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5254.
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Mouse double minute (MDM)2 is associated with proliferative vitreoretinopathy (PVR) and vitreous-induced increase in MDM2 enhances degradation of the tumor suppressor p53. The goal of this study is to use CRISPR-Cas9 to block vitreous-induced expression of MDM2 to prevent cellular responses intrinsic to PVR.
An inactive nuclease Cas9 was created by generating two point mutations in S. pyogenes Cas9(SpCas9) in a lenti-cripsr v2 vector (Addgene: 52961) using a mutagenesis kit. A previously demonstrated sgRNA targeting the first intron of MDM2 and a control sgRNA targeting lacZ gene were used. The lentivirus, generated using 293T cells, was used to infect human retinal pigment epithelial cells (ARPE-19). Genomic DNA from infected ARPE-19s was extracted using the Wizard genomic DNA purification Kit for Sanger sequencing. Expression levels of Akt, p-Akt, MDM2 and p53 were determined by western blot. A collagen gen contraction assay was performed to investigate the functional change of ARPE-19 in response to vitreous stimulation.
Sanger DNA sequencing analysis demonstrated that the mutant dSpCas9 was created and the dSpCas9 with the MDM2 sgRNA did not generate mutants surrounding or within the sgRNA protospacer. Western blot revealed a decrease in MDM2 and an increase in p53 in the cells with MDM2 sgRNA and dCas9 compared to those with the lacZ sgRNA and dCas9 prevented vitreous-induced in increase in MDM2. A contraction assay showed decreased contraction in the cells with dSpCas9 and MDM2 sgRNA compared with those with dCas9 and lacZ sgRNA.
Inactive Cas 9 targeted to the MDM2 intron is a potential therapeutic for preventing PVR.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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