The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells

Abstract

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether 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 using dual adeno-associated virus-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 adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR. Proliferative vitreoretinopathy (PVR)3 is a vision-threatening disease resulting from surgical correction of rhegmatogenous retinal detachment and open ocular injury (1), and it is characterized by the formation of preretinal or epiretinal membranes (2). The epiretinal membranes consist of extracellular matrix proteins and cells, including retinal pigment epithelial (RPE) cells, retinal glial cells, fibroblasts, and macrophages. PVR occurs in 8 -10% of patients who have undergone a surgical repair of rhegmatogenous retinal detachment and accounts for ϳ75% of all primary failures following the surgery (2-8).The oncogene protein murine double minute 2 (MDM2) is an E3 ubiquitin protein ligase whose human homologue (also called Hdm2) is an important negative regulator of the p53 tumor suppressor (9 -11). The phenotype of murine embryonic lethality of MDM2 null can be prevented by knocking out the p53 gene (12, 13). Vitreous from experimental rabbits preferentially activates platelet-derived growth factor receptor (PDGFR). This activation in turn triggers the downstream signaling pathway of PI3K/Akt, which phosphorylates MDM2, thereby enhancing p53 degradation (14). Blocking MDM2 binding to p53 with a small molecule, Nutlin-3, protects rabbits against retinal detachment in a PVR rabbit model (3).Intriguingly, the G allele of SNPs (rs2279744) in the MDM2 promoter locus has subsequently been found to be associated with a higher risk of PVR for rhegmatogenous retinal detachment patients (2, 15). This SNP is also associated with an increased risk of carcinogenesis (15-21). The SNP T309G (a T-to-G change at the 309th nucleotide) at the MDM2 first intron promoter locus enhances the affinity of the transcriptional activator specificity protein 1 (Sp1), leading to a heightened expression of MDM2 and the sub...

Genome editing abrogates angiogenesis in vivo

“…To identify an appropriate AAV serotype that could transduce vascular endothelial cells (ECs), we replaced the GFP promoter (phSyn) in the AAV-SpGuide vector 16 with a promoter of cytomegalovirus (CMV) (Fig. 1a) 15 .

Fig. 1AAV-CRISPR/Cas9-mediated depletion of VEGFR2 in vitro.

…”

CRISPR Knockdown of Kcnq3 Attenuates the M-current in NPY/AgRP Neurons

“…However, due to the large size of Staphylococccus pyogenes, there is insufficient capacity in the expression cassette of adeno-associated viruses to also include the single guide RNA (sgRNA) necessary for gene targeting. This limitation requires use of a separate viral vector [50,51] or incorporation of a Cre-dependent SpCas9 transgenic line [52]. We recently developed a single viral vector for conditional expression of the smaller Staphylococcus aureus (SaCas9) and sgRNA that yields high-efficiency mutagenesis in specific cell types [41].…”

Progress in the application of CRISPR: From gene to base editing

“…To convert A·T to G·C in genomic DNA, in theory deamination of adenosine to inosine that is treated as G by polymerase could be used to develop such a system of adenine base editors (ABEs). Although there is no enzyme found to deaminate adenine in DNA, a transfer RNA (tRNA) adenine deaminase (TadA) can convert A to I in the single‐stranded anticodon loop of tRNA. After failure to establish ABEs by following the construction of BE system, the Liu’s group has developed a series of ABEs based on E. coli TadA (ecTadA) for switching A·T to G·C in genomic DNA (Figure ).…”

“…NHEJ introduces insertions or deletions (indels) at the damage site, causing frame shifts in coding sequences and subsequent protein depletion . In the presence of a homologous recombination template, Cas9‐directed HDR can correct poisonous mutations . This traditional CRISPR/Cas9 strategy depends on induction of a DNA break, which may create undesired consequences.…”