Figure 6. Analyses of targeted mutagenesis in human myoblasts following AdV-mediated transfer of DNA coding for RGN and TALEN complexes.

(A) Targeted mutagenesis experiments with AdV^Δ2P.Cas9.F⁵⁰ and AdV^Δ2U6.gRNA^S1.F⁵⁰. Upper panel, AAVS1-specific PCR products corresponding to genomic DNA of myoblasts co-transduced with the indicated MOIs of AdV^Δ2P.Cas9.F⁵⁰ and AdV^Δ2U6.gRNA^S1.F⁵⁰ (in TU/cell) subjected to T7EI-based assays. Lower panel, negative controls corresponding to human myoblasts that were singly transduced with AdV^Δ2P.Cas9.F⁵⁰ (lane 1), AdV^Δ2U6.gRNA^S1.F⁵⁰ (lane 2) or AdV^Δ2P.TALEN-L^S1.F⁵⁰ (lane 3) or that were mock-transduced (lane 4). (B) Target gene knockout frequencies in human myoblasts co-transduced with AdV^Δ2P.Cas9.F⁵⁰ and AdV^Δ2U6.gRNA^S1.F⁵⁰ as measured by densitometry of the DNA species depicted in Fig. 6A. (C) Targeted mutagenesis experiments with AdV^Δ2P.TALEN-L^S1.F⁵⁰ and AdV^Δ2P.TALEN-R^S1.F⁵⁰. Upper panel, T7EI-based assays on AAVS1-specific amplicons obtained from genomic DNA of myoblasts co-transduced with the indicated MOIs of AdV^Δ2P.TALEN-L^S1.F⁵⁰ and AdV^Δ2P.TALEN-R^S1.F⁵⁰ (in TU/cell). Solid and open arrowheads indicate the positions of undigested and T7EI-digested DNA molecules, respectively. Lanes M, Gene Ruler DNA Ladder marker. Lower panel, target gene disruption frequencies in myoblasts co-transduced with AdV^Δ2P.TALEN-L^S1.F⁵⁰ and AdV^Δ2P.TALEN-R^S1.F⁵⁰ as determined by densitometry of the DNA fragments shown in the upper panel.