The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells

Abstract

The 309G allele of single nucleotide polymorphisms (SNPs) in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but as to whether this SNP G309 contributes to the pathogenesis of PVR is to-date unknown. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here, we delivered SpCas9 and guide RNAs (SpGuides) using dual adeno-associated viral (AAV)-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells, whose genotype is MDM2 T309T. The next generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using the AAV-CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in the MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that the MDM2 T309G in the hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.