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. 2016 May 31;291(31):16339–16347. doi: 10.1074/jbc.M116.729467

FIGURE 2.

FIGURE 2.

Identification of MDM2-sgRNAs. A, the genomic DNA was isolated from hPRPE cells and subjected to PCR amplification of a region around the MDM2 SNP 309. The PCR products were purified from a 2% agarose gel for DNA sequencing. B, graphic representation of the MDM2-targeted loci. 20-nt target 1 (612–631) and target 2 (583–675) in the human genomic MDM2 loci (NC_000012.12) are indicated by a green arrow for nicking at sites (red arrows) by SpCas9D10A. The protospacer-adjacent motifs (PAM) are marked in blue. C, genomic DNA was extracted from transfected cells using the QuickExtract DNA extraction solution system following the protocol of the manufacturer for amplification of a region around MDM2 SNP 309. The gel-purified PCR products were subjected to DNA sequencing analysis. MDM2-sgRNA1 or 2 or LacZ-sgRNA, partial sequencing from the DNA extracted from the transfected cells with pAAV-SpCas9 and pAAV-MDM2-sgRNA1 or 2 (or LacZ-sgRNA)-CMV-GFP. Arrows indicate the expected SpCas9 cleavage sites.