The crispr / cas 9-created mdm 2 t 309 g enhances vitreous-induced expression of mdm 2 and proliferation and survival of cells

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 adenoassociated 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. Proliferative vitreoretinopathy (PVR) is a vision-threatening disease resulting from surgical correction of rhegmatogenous retinal detachment (RRD) and open ocular injury (1), and it is characterized by the formation of preretinal or epiretinal membranes (ERMs) (2). The ERMs consist of extracellular MDM2 T309G enhances vitreous-induced cell survival 2 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 RRD, and accounts for approximately 75% of all primary failures following the surgery (28). The oncogene protein murine double minute 2 (MDM2), an E3 ubiquitinprotein 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 plateletderived growth factor receptor α (PDGFRα). This activation in turn triggers the downstream signaling pathway of phosphoinositide 3 kinase (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 single nucleotide polymorphisms (SNPs) (rs2279744) in the MDM2 promoter locus has subsequently been found to be associated with a higher risk of PVR for RRD 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 309 nucleotide) at the MDM2 first-intron promoter locus enhances the affinity of the transcriptional activator specificity protein (Sp)1, leading to a heightened expression of MDM2 and the subsequent attenuation of p53 expression in cancer cells (15). However, whether or not this SNP contributes to the pathogenesis of PVR has not been explored. The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nucleases (Cas) in bacteria and archaea provides adaptive immunity against viruses and plasmids when their CRISPR RNAs (crRNAs) are used to guide the Cas cleavage of the foreign nucleic acids (2224). In Streptococcus pyogenes (Sp) the Cas9 (SpCas9) contains two nuclease domains, RuvC and HNH, each of which can cleave one strand of the doublestranded target DNA when directed by the crRNA and transactivating crRNA (tracrRNA) (24,25). This SpCas9 can be reprogrammed to target specific genomic loci in mammalian cells using the processed single guide (sg) RNAs that consist of the crRNA and tracrRNA(24). The double-stranded DNA breaks (DSBs), at the specific genomic loci produced by the CRISPR/Cas9, can be repaired by endogenous repair machinery for either non-homologous end-joining (NHEJ) or homology-directed repair (HDR), which depends on the cell state and presence of a repair template (26,27). NHEJ and HDR are two distinct competent repair pathways in the cells. NHEJ can introduce unpredictable insertions and deletions (indels), and it may repair the lesion by simply rejoining the two DSB ends (26); HDR can use an exogenous singleor double-stranded DNA template with the desired changes to make mutations in the genomic loci (26). However, HDR is less frequently used than NHEJ because it occurs only during S and G2 phases, whereas NHEJ can be found throughout the cell cycle (26,28). The CRISPR/Cas9 technology has recently been used in a variety of genome-editing applications in eukaryotic cells and mice (26,29-32), and it provides a unique opportunity to demonstrate whether the MDM2 T309G contributes to the pathogenesis of PVR. MDM2 T309G enhances vitreous-induced cell survival 3 Here, we generated the mutation of T309G in the MDM2 genomic locus, in the human primary retina pigment epithelial (hPRPE) cells using CRISPR/Cas9 technology. We demonstrated that the vitreous from experimental rabbits (RV) increased expression of MDM2 and subsequent attenuation of p53 expression in the hPRPE cells with the MDM2 T309G; furthermore, we found that the MDM2 T309G in the hPRPE cells promoted RV-induced cell proliferation and survival, which are intrinsic to the development of PVR. RESULTS Creation of MDM2 T309G in the genomic locus using CRISPR/Cas9 − The SNP MDM2 T309G is associated with a higher risk of PVR (2)(2); however, it is not known whether this SNP contributes to PVR. Because RPE cells are believed to be the major cell type in the PVR membranes that cause retinal detachment in the development of PVR (2,5-7), we attempted to create this SNP in hPRPE cells using CRISPR/Cas9 technology. Since the ultimate goal of this research is to explore a novel therapeutic approach to PVR, and AAVs do not cause any disease (33), we chose AAV-derived viral vectors to deliver CRISPR/Cas9 into our target cells. However, due to the packaging size limitation of the AAV-derived vectors, we had to adapt a dual-vector system, which packages SpCas9 and sgRNA expression cassettes (SpGuide) in two separate viral vectors, pAAV-SpCas9 and pAAVSpGuide, respectively (30). To separate hPRPE cells transduced by pAAVSpGuide, we replaced GFP promoter hSyn (30) with the promoter of CMV (Fig. 1A). There are four types of cells in the PVR membrane, including RPE cells; therefore, we substituted the promoter pMecp2 (30) for the promoter of the RSV to drive expression of SpCas9 (34) (Fig. 1B). The SpCas9 contains two conserved nuclease domains, HNH and RuvC, which cleave the target DNA strand complementary and non-complementary to the guide RNA, respectively. A mutation of aspartate-to-alanine (D10A) in the RuvC catalytic domain can convert SpCas9 into the DNA nickase (SpCas9D10A). Two SpCas9 D10Anicking enzymes directed by a pair of sgRNAs targeting opposite strands of a target locus can mediate double DNA strand breaks while minimizing off-target activity, because single-strand nicks are preferentially repaired by the high-fidelity base excision repair pathway (35). Thus, the SpCas9 in the AAV vector was mutated to SpCas9 D10A with an in situ mutagenesis kit (Fig. 1B). To test the effectiveness of the DNA constructs, we transfected these two vectors separately into 293T cells. Western blot analysis showed that the SpCas9 and SpCas9 D10A were successfully expressed in the transfected 293T cells (Fig. 1C). We next sought to test the efficiency of SpCas9-mediated editing of the genomic MDM2 locus around the SNP in the RPE cells. To identify RPE cells that were suitable for our experimental purpose, we isolated the genomic DNA from hPRPE cells and PCR amplified a region around the MDM2 SNP for Sanger DNA sequencing, and found that there was T309T in the MDM2 SNP in the hPRPE cells as shown in Fig. 2A. These cells were then used to introduce MDM2 T309G in the genomic locus. In synthesizing sgRNAs the two 20-nt targeted sequences (30,36-38) (Fig. 2B) were cloned into the pAAV-SpGuide backbone (Fig. 1A), and the clones were verified by DNA sequencing. MDM2 T309G enhances vitreous-induced cell survival 4 To edit the genomic MDM2 locus, we transfected hPRPE cells with the dual vectors of pAAV-SpCas9 plus pAAVMDM2-sgRNA 1 or 2 using electroporation. pAAV-LacZ-sgRNA was used as a control. The transduction efficiency was about 60%, as estimated by immunofluorescence. To determine if any indels were mediated by the CRISPR/Cas9 system, we isolated genomic DNA from the transfected hPRPEs and amplified the region around the MDM2 SNP using a high fidelity Herculase II fusion polymerase. The amplified DNA fragments were then subjected to Sanger DNA sequencing (39). As shown in Fig. 2C there were mutations in front of PAMs (MDM2-sgNRA1: CGG; MDM2-sgRNA2: AGG) from the PCR products derived from the transduced hPRPE cells with SpCas9 plus MDM2-sgRNA 1 or 2, but not from those with LacZ-sgRNA. These results demonstrate that the two MDM2 sgRNAs efficiently guided the SpCas9 to induce indels in the hPRPE cells. To create the SNP MDM2 T309G in the genome of the hPRPE cells, we chose to use SpCas9D10A nickase activity, as there was no off-target DNA sequence found for the pair of sgRNA1 +2, based on the double nickase design tool (crispr.mit.edu). Thus, the U6-sgRNA2 was PCR amplified and cloned into the pAAV-U6-sgRNA1 vector. The pAAVSpCas9D10A, pAAV-MDM2-sgRNA 1 and 2 and ssHRT (Fig. 3A) together were transfected into the hPRPE cells by electroporation. The HDR donor template that consisted of a single strand, 96 base pair (bp) genomic sequence homologous to a region encompassing the SNP with a G309 re