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. 2014 Mar 5;9(3):e89413. doi: 10.1371/journal.pone.0089413

Figure 3. Cas9:sgRNA-mediated modifications in 4 genes by a mixture of dual sgRNAs for each gene.

Figure 3

(a) PCR identification of sgRNA:Cas9-mediated site-specific cleavage of the endogenous ApoE, B2m, Prf1, and Prkdc loci. The genetic modification analysis was performed by PCR amplification of the targeted fragment in the ApoE, B2m, Prf1, and Prkdc in 25 founder rats (#16∼40) derived from co-microinjection of a mixture of dual sgRNAs for each genes as described in Table S2 in File S1. Primer used for PCR amplication was described in Table S3 in File S1. Additionally, founder #36, #38 had a larger deletion in the ApoE, the primer ApoE-NS2 and ApoE-NAS2 used for amplification. Founder #38 had a larger deletion in the B2m, the primer B2m-S2 and B2m-AS2 used for amplification. (b) Detection of Cas9:sgRNA-mediated site-specific cleavage of the endogenous ApoE, B2m, Prf1, and Prkdc by T7EN1 cleavage assay. PCR products from (a) were subjected to T7EN1 cleavage assay as described in material and methods.