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The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created

Author(s)
Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A.; Zhang, Feng; Lei, Hetian; ... Show more Show less
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Alternative title
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
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Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/
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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.
Date issued
2016-03
URI
http://hdl.handle.net/1721.1/113194
Department
Broad Institute of MIT and Harvard; Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry and Molecular Biology (ASBMB)
Citation
Duan, Yajian et al. “The Clustered, Regularly Interspaced, Short Palindromic Repeats-Associated Endonuclease 9 (CRISPR/Cas9)-createdMDM2T309G Mutation Enhances Vitreous-Induced Expression of MDM2 and Proliferation and Survival of Cells.” Journal of Biological Chemistry 291, 31 (May 2016): 16339–16347 © 2016 The American Society for Biochemistry and Molecular Biology, Inc
Version: Original manuscript
ISSN
0021-9258
1083-351X

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