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Adenoviral vector DNA for accurate genome editing with engineered nucleases

Abstract

Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator–like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.

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Figure 1: Gene targeting with IDLV donor DNA and TALEN-induced DSBs.
Figure 2: Gene targeting with AdV donor DNA and TALEN-induced DSBs.
Figure 3: Nuclease-mediated gene targeting of AdV donor DNA in genetically unstable HeLa cells.
Figure 4: Nuclease-induced gene targeting with AdV- versus plasmid-mediated delivery of HR substrates.
Figure 5: The protein cap of AdVs contributes to gene-targeting specificity and accuracy.

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Acknowledgements

The authors thank J. Liu for her technical assistance and L.P. Pelascini for the generation of the lentiviral vector stocks encoding ZFNs. We also thank A. Recchia (University of Modena and Reggio Emilia) for providing us with plasmid pSh.AAVS1.eGFP; R. Hoeben and D. Baker for critically reading the manuscript; K. Szuhai and D. de Jong for the COBRA-FISH karyotyping; and M. Rabelink for the p24gag ELISA measurements. This work was supported by the Prinses Beatrix Spierfonds (grant W.OR11-18 to M.A.F.V.G.), the European Community's 7th Framework Programme for Research and Technological Development (PERSIST grant 222878 to T.C. and M.A.F.V.G.) and the European Community's ERASMUS Programme (grant PLISBOA02 to S.F.D.H.).

Author information

Authors and Affiliations

Authors

Contributions

M.H. and I.M. contributed equally to this work. M.H. and I.M. generated reagents and performed most of the experiments with the help of S.F.D.H., J.M.J. and M.A.F.V.G. T.C. generated and validated AAVS1-specific TALENs and EGFP-specific ZFNs. M.H., I.M., J.M.J. and M.A.F.V.G. designed the experiments and analyzed the data. M.A.F.V.G. conceived of and initiated the research. M.A.F.V.G. wrote the manuscript with the help from all authors.

Corresponding author

Correspondence to Manuel A F V Gonçalves.

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Competing interests

M.H. and M.A.F.V.G. are co-inventors on a filed patent application covering findings described in the present study.

Integrated supplementary information

Supplementary Figure 1 Characterization of human myoblasts genetically modified by using TALENs and IDLV donor DNA.

(a) Two-color direct fluorescence microscopy on myoblast cultures treated with TALENs and IDLV donor DNA. Target cells were transduced with the EGFP-encoding IDLV.donorS1 (+, -) or were co-transduced with IDLV.donorS1 and with AdV.TALEN-LS1 alone (+, L) or mixed with AdV.TALEN-RS1 (+, L, R). Owing to a bi-cistronic expression unit, cells transduced with the TALEN-encoding AdVs become tagged with the DsRedEx2.1 reporter. (b) Gene targeting analyses by PCR screening of EGFP+ myoblast clones derived from cultures exposed to AAVS1-specifc TALENs and IDLV.donorS1. Upper panel, diagram of PCR assays for detecting HR-derived “telomeric” (jT) and “centromeric” (jC) IDLV DNA-AAVS1 junctions and head-to-tail IDLV DNA concatemers (jH-T). PCR amplifications targeting EGFP were used as internal controls for the presence and integrity of the chromosomal DNA (EGFP). Lower panels, PCR screening of randomly selected EGFP+ myoblast clones by using the PCR assays depicted in the upper panel. Open arrowheads, IDLV integrants lacking HR-derived exogenous DNA-AAVS1 junctions at either one or both termini; solid arrows, IDLV integrants comprising head-to-tail concatemers. Nuclease-free water and chromosomal DNA of unmodified myoblasts (Mock) provided for negative controls, whilst DNA extracted from sorted EGFP+ myoblasts served as positive controls (pool). The PCR screening results are compiled in the plot of Fig. 1c. (c) Examining nuclease-independent IDLV gene targeting. PCR analysis of chromosomal DNA from sorted EGFP+ myoblasts transduced with IDLV.donorS1 in the absence of TALEN gene expression (IDLV+ TALEN-) to probe for the formation of “telomeric” (jT) and “centromeric” (jC) exogenous DNA-AAVS1 junctions. Nuclease-free water and chromosomal DNA of mock-transduced myoblasts served as negative controls, whilst DNA extracted from an AAVS1-targeted EGFP+ myoblast clone provided for positive controls. PCR amplifications targeting EGFP were used as internal controls. Marker, GeneRuler DNA Ladder Mix molecular weight marker.

Supplementary Figure 2 Schematic representation of exogenous DNA forms found in myoblasts genetically modified by using TALENs and IDLV donor DNA.

(a) Diagram of AAVS1 target site and IDLV.donorS1. Magenta boxes, first, second and third exons of PPP1R12C; ψ, HIV-1 packaging signal; white and grey boxes, 5′ and 3′ LTR, respectively. The transgene in IDLV.donorS1 contains the human PGK-1 promoter controlling EGFP expression; half arrows, target site-specific primers. (b) Overview and nomenclature of IDLV.donorS1 arrangements identified by PCR screening of EGFP+ myoblast clones retrieved from populations co-transduced with AdV.TALEN-LS1 and AdV.TALEN-RS1. From this analysis it resulted that, in addition to integrants displaying bona fide HR-derived “telomeric” and “centromeric” AAVS1-exogeous DNA junctions (class I), there were also those lacking either one (classes IIa and IIb ) or both (class III) of these junctions. Moreover, clones consistent with the aforementioned classes and displaying, in addition, a direct repeat concatemeric arrangement could also be identified (classes Ic, IIac, IIbc and IIIc). jT, jC and jH-T, amplicons diagnostic for “telomeric”, “centromeric” and head-to-tail IDLV DNA junctions, respectively.

Supplementary Figure 3 Gene targeting with IDLV donor DNA and ZFN-induced DSBs.

(a) The ZFNs composed of zinc-finger arrays fused to the FokI nuclease domain are drawn in relation to their cognate target sites present within EGFP. These bipartite sequences frame the spacer elements (lower case) whose sequences are cleaved upon the local assembly of nuclease pairs and ensuing dimerization-dependent FokI catalysis. IDLV.donorEGFP genomes contain as HR substrate an expression unit flanked by sequences identical to those bracketing the ZFN target site. The expression unit consists of the hybrid CAG promoter, the FP635 (a.k.a. katushka) reporter gene and the bovine GH-1 polyadenylation signal. EF1α promoter, human EEF1A1 regulatory sequences; SUR, SV40 5′UTR (SU) and R region (R) from HTLV-1; EGFP, reporter-encoding ORF; GH pA, human GH-1 polyadenylation signal; Ψ, HIV-1 packaging signal; white and grey boxes, 5′ and 3′ retroviral long terminal repeat sequences, respectively. (b) IDLV gene targeting in indicator H27 cells. Cells were transduced with IDLV.donorEGFP (-) or with LV.ZFN-1EGFP and LV.ZFN-2EGFP (+). Flow cytometry was performed at 35 days post-transduction. H27 cells containing EGFP-targeted exogenous DNA or with NHEJ-disrupted EGFP plus random exogenous DNA insertion acquire an EGFP- FP635+ phenotype (red bar). H27 cells subjected exclusively to off-target insertions become marked as EGFP+ FP635+ (orange bars).

Supplementary Figure 4 Molecular characterization of myoblasts genetically modified by deploying TALENs and AdV donor DNA.

(a) Gene targeting analyses by PCR screening of EGFP+ myoblasts derived from cultures exposed to AAVS1-specifc TALENs and AdV.Δ2.donorS1. PCR assays specific for HR-derived “telomeric” (jT) and “centromeric” (jC) AdV DNA-AAVS1 junctions were performed on DNA isolated from randomly selected EGFP+ myoblast clones. PCR amplifications targeting EGFP served as internal controls for the presence and integrity of the chromosomal DNA (EGFP). Nuclease-free water and DNA from mock-transduced myoblasts were used as negative controls, whereas DNA from IDLV.donorS1-targeted myoblast clone 4 (IDLV.4) and plasmid pAdV.donorS1 (shuttle) provided for positive controls. These PCR screening data are collected in the graph of Fig. 2d. (b) Probing by PCR for the presence of head-to-tail AdV DNA concatemers in stably transduced myoblasts. Left panel, schematics of in vitro-assembled head-to-tail AdV DNA junctions (jH-T). L-ITR and R-ITR, “left” and “right” AdV ITR, respectively; half arrows, primers; horizontal bar, amplicon diagnostic for the presence of head-to-tail AdV DNA concatemers. Right panel, PCR analysis of chromosomal DNA from EGFP+ myoblasts treated with AAVS1-specific TALENs and AdV.Δ2.donorS1 (gDNA EGFP+ pool). DNA from parental myoblasts (gDNA myoblasts) and nuclease-free water provided for negative controls, whereas in vitro-generated head-to-tail AdV DNA templates (left panel) constituted the positive control of this assay (H-T AdV DNA). PCR amplifications targeting EGFP were used as internal controls (EGFP). Marker, GeneRuler DNA Ladder Mix molecular weight marker.

Supplementary Figure 5 Southern blot analyses of human myoblasts stably transduced following AdV-mediated delivery of TALENs and donor DNA.

(a) Southern blot analysis of EGFP+ myoblast clones. NcoI-digested genomic DNA was resolved through agarose gels, blotted and incubated with an AAVS1-specific probe (short horizontal black bar). Left-hand panel, autoradiogram showing myoblast clones displaying the typical chromosomal integration pattern of AdV-delivered donor DNA resulting from HR events at AAVS1 (solid arrowhead). Middle panel, autoradiogram displaying the DNA from myoblast clone 10 subjected to bi-allelic HR-mediated gene targeting at AAVS1 loci (vertical open arrow). Right-hand panel, autoradiogram exhibiting the DNA corresponding to myoblast clone 58 whose restriction pattern is consistent with an additional, HR-independent, insertion event presumably brought about by the incorporation of AdV.Δ2.donorS1 backbone sequences (horizontal open arrow). Lanes M, GeneRuler DNA Ladder Mix molecular weight marker. Lane C, Genomic DNA from unmodified human myoblasts. (b) Southern blot analysis of the parental EGFP+ myoblast population. NcoI-digested DNA was Southern blotted and incubated with an EGFP-specific probe (short horizontal green bar). The autoradiogram correspond to genomic DNA of myoblast clone 88 and to genomic DNA (10 μg and 20 μg) of the EGFP+ myoblast population from which the clones were isolated from. The position of radiolabelled DNA fragments diagnostic for HR-mediated chromosomal integration of transgenic sequences at AAVS1 is indicated (solid arrowhead). Marker, GeneRuler DNA Ladder Mix molecular weight marker.

Supplementary Figure 6 COBRA-FISH karyotyping of HeLa target cells highlighting the high genomic DNA instability of these human cervix carcinoma cells.

Supplementary Figure 7 Molecular characterization of HeLa cells genetically modified by deploying TALENs and AdV donor DNA.

Gene targeting analyses by PCR screening of EGFP+ HeLa cell clones expanded from cultures exposed to AAVS1-specifc TALENs and AdV.Δ1.donorS1. PCR assays specific for HR-derived “telomeric” (jT) and “centromeric” (jC) AdV DNA-AAVS1 junctions were performed on chromosomal DNA from randomly selected EGFP+ HeLa cell clones. PCR amplifications targeting EGFP served as internal controls for the presence and integrity of DNA templates (EGFP). Marker, GeneRuler DNA Ladder Mix molecular weight marker. DNA from mock-transduced HeLa cells, sorted EGFP+ HeLa cells (pool) and EGFP+/donor DNA- H27 cells served as controls in these PCR assays. The gathered data is plotted in the graph of Fig. 3d.

Supplementary Figure 8 Genotyping assay for validating AdV.Cas9 and AdV.gRNAS1 in HeLa cells.

PCR products spanning the AAVS1 target region (Fig. 4a) were amplified from genomic DNA of HeLa cells transduced with AdV.Cas9 alone at an MOI of 300 TCID50/cell (Cas9) or with 1:1 mixtures of AdV.Cas9 and AdV.gRNAS1 at a total MOI of 60, 120, 180, 240 and 300 TCID50/cell. After amplicon denaturation/re-annealing, base pair mismatches (indels) resulting from NHEJ-mediated DSB repair were detected by T7 endonucleases I digestions (+T7EI). Amplicons not exposed to T7EI provided for negative controls (-T7EI). Solid and open arrowheads indicate the positions of, respectively, undigested and T7EI-digested DNA fragments.

Supplementary Figure 9 Designer nuclease–induced genetic modification of target cells following delivery of HR substrates in plasmid versus AdV DNA.

HeLa cells were either co-transduced with AdV.Cas9, AdV.gRNAS1 and AdV.Δ2.donorS1 (first panel) or were co-transfected with the plasmid pair encoding the AAVS1-specific TALENs plus PacI-linearized pAdV.donorS1, covalently-closed pAdV.donorS1/T-TS or covalently-closed pAdV.donorS1 (second, third and fourth panel, respectively). Parallel HeLa cell cultures that were co-transduced with AdV.Cas9 and AdV.Δ2.donorS1 or that were co-transfected with the TALEN-LS1-encoding expression construct mixed with each of the three donor plasmid types provided for negative controls. The frequency of EGFP+ cells present in the various long-term HeLa cell cultures was determined by flow cytometry at 23 days after transgene delivery and is indicated within each dot plot. Pictograms of the various types of donor DNA templates deployed in these experiments are drawn next to their respective dot plots. Thin parallel lines, double-stranded DNA; Ψ, AdV packaging signal; horizontal black, white and grey bars, donorS1 DNA composed of AAVS1 targeting sequences framing the EGFP-encoding transgene (Exo.); TP, terminal protein covalently attached at the 5′ termini of the AdV DNA; vertical arrowheads, position of the recognition sequences for the restriction enzyme PacI and the designer nuclease dimer complex TALEN-LS1:TALEN-RS1; ori and KanR, prokaryotic origin of replication and kanamycin-resistance gene, respectively. DSB, double-stranded DNA break.

Supplementary Figure 10 Transgene expression profiles determined by flow cytometric analyses of genetically modified HeLa cells.

(a) Relationship between the CV and MFI values on a per clone basis. The various EGFP+ HeLa cell clones were generated by using the AAVS1-specific TALENs plus covalently-closed pAdV.donorS1/T-TS (first graph), PacI-linearized pAdV.donorS1 (second graph) or covalently-closed pAdV.donorS1 (third graph) or were made instead by deploying the AAVS1-specific RGN complex plus protein-capped AdV.Δ2.donorS1 DNA (fourth graph). Plotting the relationship between the CV and the MFI parameters for each individual clone (colored solid circles) highlights the higher homogeneity of transgene expression found in the clonal set modified with AdV donor DNA. (b) Transgene expression profiles of genetically modified HeLa cell populations. The different EGFP+ HeLa cell populations were generated by applying AAVS1-specific TALENs or RGN complexes together with linear free-ended HR substrates generated by PacI digestion of pAdV.donorS1 (orange histograms) or by designer nuclease-mediated cutting of pAdV.donorS1/T-TS (magenta histograms). Of note, the donor DNA-framing T-TS sequences in pAdV.donorS1/T-TS contain the recognition site for Cas9:gRNAS1 complexes. As a result, pAdV.donorS1/T-TS is susceptible to not only AAVS1-specific TALENs but also to AAVS1-specifc RGN complexes. For the sake of comparison, the transgene expression profile corresponding to the EGFP+ HeLa cell population obtained after co-delivering Cas9:gRNAS1 complexes and AdV.Δ2.donorS1 DNA is also shown (green histograms). This analysis confirms that, when compared to the deployment of AdV donor DNA, the use of free-ended plasmid donor DNA leads to higher heterogeneity of transgene expression regardless of the designer nuclease platform chosen for site-specific DSB formation.

Supplementary Figure 11 Gene-targeting analyses of HeLa cells genetically modified by using designer nucleases and different types of HR substrates.

(a) Analysis of HR-mediated gene targeting of in cellula-linearized donor DNA. PCR screening of EGFP+ HeLa cell clones isolated from cultures subjected to the transfer of AAVS1-specific TALENs and pAdV.donorS1/T-TS. Targeted gene addition and intermolecular donor DNA recombination events were assessed by PCR amplifications specific for “telomeric” AAVS1-exogenous DNA junctions (jT) and head-to-tail exogenous DNA concatemers (jH-T), respectively. (b) Analysis of HR-mediated gene targeting of covalently-closed circular donor DNA. PCR screening of EGFP+ HeLa cell clones isolated from cultures subjected to the transfer of AAVS1-specific TALENs and pAdV.donorS1. (c) Analysis of HR-mediated gene targeting of protein-capped linear donor DNA. PCR screening of EGFP+ HeLa cell clones derived from cultures exposed to Cas9:gRNAS1 and AdV.Δ2.donorS1. In all samples, PCR amplifications targeting EGFP served as internal controls for the presence and integrity of DNA templates. Vertical arrowheads indicate clones lacking the targeted exogenous DNA-AAVS1 junction. Nuclease-free water and genomic DNA of mock-transduced HeLa cells served as negative controls, whilst genomic DNA extracted from an AAVS1-targeted EGFP+ HeLa cell clone served as positive controls for the EGFP- and “telomeric” junction (jT)-specific PCR amplifications. The PCR screening designed to assess head-to-tail donorS1/T-TS concatemer formation used, as a positive control, genomic DNA extracted from the respective parental EGFP+ population. Ψ, adenoviral packaging signal; TP, adenoviral terminal protein. Lanes M, GeneRuler DNA Ladder Mix molecular weight marker. These cumulative data are gathered in Fig. 4d.

Supplementary Figure 12 Prokaryotic DNA status of cell populations genome edited by combining engineered nucleases with AdV or plasmid HR templates.

KanR-specific PCR analysis of genomic DNA extracted from EGFP+ sorted HeLa cells initially transduced with AdV.Δ2.donorS1 (AdV DNA) or transfected with pAdV.donorS1 (supercoiled), PacI-linearized pAdV.donorS1 (linear in vitro) or TALEN pair-susceptible pAdV.donorS1/T-TS (linear in vivo). Targeted DSBs were induced in AdV-transduced and plasmid-transfected cells by using AAVS1-specific RGN (Cas9:gRNAS1) and TALEN (TALENS1 [L/R]) complexes, respectively. Plasmid pAdV.donorS1 served as positive control (+), whereas genomic DNA from parental HeLa cells (-) and nuclease-free water provided for negative controls. The integrity of the various DNA templates was controlled for by carrying out parallel EGFP-specific PCR amplifications. Lane M, GeneRuler DNA Ladder Mix molecular weight marker.

Supplementary Figure 13 Testing TALEN-mediated release of donor DNA from AdV genomes in transduced cells.

(a) Characterization of control AdV.Δ2.donorS1/FRT and test AdV.donorS1/T-TS DNA by restriction fragment length analysis. Left-hand panel, generic structures of AdV.Δ2.donorS1/FRT and AdV.Δ2.donorS1/T-TS recombinant genomes. FRT and T-TS, target sites for the yeast site-specific FLP recombinase and the AAVS1-specific TALEN pair, respectively; open box, exogenous DNA flanked by AAVS1-targeting sequences (black and grey bars); Ψ, AdV packaging signal; open oval, 5′ covalently-attached AdV terminal protein. Central and right-hand panels, in silico and actual electrophoretic mobility pattern of restriction fragments upon treatment of control and test vector DNA with XbaI and MluI, respectively. The in silico XbaI and MluI restriction patterns, made by using the Gene Construction Kit (version 2.5), aided in establishing the integrity of vector DNA templates. Marker, GeneRuler DNA Ladder Mix molecular weight marker. (b) Experimental design. Cellular DNA was extracted from HeLa cells co-transduced with AdV.Δ2.donorS1/FRT and FLPe-encoding vector hcAd.FLPe.F50 and from HeLa cells transduced with a combination of AdV.Δ2.donorS1/T-TS, AdV.Δ2.TALEN-LS1 and AdV.Δ2.TALEN-RS1 (L/R). PCR amplifications with “outward-facing” primers (half arrows) on linear templates will result in specific products only upon donor DNA excision and circularization. From this experimental setup it follows that direct FLPe-induced excision and circularization of donor DNA from AdV.Δ2.donorS1/FRT provides for in vivo-generated positive control templates. Detection of a similarly sized PCR species diagnostic for NHEJ-mediated circularization of linear donor DNA serves as a surrogate indicator for TALEN-dependent excision of donor DNA from AdV.donorS1/T-TS genomes. The size and position (in kb) of these PCR species are indicated on the right side of the agarose gel.

Supplementary Figure 14 Live-cell tracing of genome modification events in H27 cells following the delivery of TALENs and AdV donor DNA.

(a) Diagram of AdVs used in these experiments. AdV.TALEN-LEGFP and AdV.TALEN-REGFP encode TALENs addressed to an EGFP sequence, whereas AdV.Δ2.donorEGFP contains EGFP target sequence-matched donor DNA coding for the far-red fluorescent protein FP635. The repeat sets of the TALEN ORFs and of the corresponding TALENs conferring DNA-binding specificity are color-coded. Each TALEN is drawn in relation to its respective half target site. Ψ, adenoviral packaging signal; Solid boxes, adenoviral inverted terminal repeats; CMV, cytomegalovirus immediate-early gene promoter. For an explanation of the other symbols and elements see the legend of Supplementary Fig. 3. (b) Two-color fluorescence microscopy analysis of mock-transduced H27 cells and of H27 cells transduced with AdV.Δ2.donorEGFP (4 TU/cell) plus AdV.TALEN-LEGFP and AdV.TALEN-REGFP at an MOI of 3×103 VP/cell each. Micrographs were taken at 32 days post-transduction. Arrows point to areas containing EGFP- FP635+ cells with every cell present in each field being identified by Hoechst 33342 staining. (c) Two-color flow cytometric analysis of H27 cells transduced with AdV.Δ2.donorEGFP (4 TU/cell) plus 1:1 mixtures of AdV.TALEN-LEGFP and AdV.TALEN-REGFP applied at total MOIs of 6×103, 2.4×103 and 9.6×102 VP/cell (L/R). Controls consisted of H27 cells, HeLa cells exposed for 3 days to AdV.Δ2.donorEGFP (0.5 TU/cell) and H27 cells transduced with AdV.Δ2.donorEGFP (4 TU/cell) plus AdV.TALEN-LEGFP at 6×103, 2.4×103 and 9.6×102 VP/cell (L). Dot plots of cultures subjected to TALEN expression were acquired at 37 days post-transduction. (d) Graph corresponding to the flow cytometry data presented in panel c. (e) Transgene expression profile of H27-derived clones. Flow cytometry histograms corresponding to the EGFP- FP635+ cell lines isolated from H27 cultures subjected to AdV.Δ2.donorEGFP transduction and to the highest combined dose of AdV.TALEN-LEGFP and AdV.TALEN-REGFP (L/R). (f) Transgene expression levels in H27-derived clones. Ranking of the MFI values corresponding to the histograms shown in panel e. (g) Gene targeting analysis by PCR screening of EGFP- FP635+ H27-derived clones. Upper panel, diagram of the PCR assay for detecting homology-directed gene targeting at EGFP. For an explanation of the symbols and elements see the legend of Supplementary Fig. 3. PCR amplifications specific for HPRT1 served to control for the presence and integrity of the genomic DNA templates. Nuclease-free water and genomic DNA of HeLa cells (Mock) provided for negative controls, whereas DNA isolated from sorted EGFP- FP635+ cells served as an additional target template (Pool).

Supplementary Figure 15 Examining illegitimate recombination of IDLV versus AdV DNA in myoblasts undergoing cell-cycle arrest and differentiation.

(a) Experimental design. Myoblasts were transduced with AdV.Δ2.donorS1 or with IDLV.donorS1 each mixed with AdV.TALEN-LS1 plus AdV.TALEN-RS1 (L, R) or with AdV.TALEN-RS1 alone (R), after which myoblast-to-myotube differentiation was triggered through the substitution of regular for mitogen-free medium. (b) PCR assays for detecting head-to-tail vector DNA forms present in the post-mitotic myotube cultures. The 372-bp amplicon diagnostic for a head-to-tail arrangement of AdV DNA could not be detected, whereas the 560-bp specific for IDLV DNA acquiring this DNA structure could be readily detected. Lanes M, GeneRuler DNA Ladder Mix molecular weight marker. Negative controls were provided by nuclease-free water and DNA extracted from mock-transduced myoblast cultures (-), whereas positive controls were obtained by using in vitro-assembled head-to-tail AdV DNA templates (Fig. 3c and Supplementary Fig. 3b) and DNA of IDLV.donorS1-transduced myoblast clone 10. The integrity of the various DNA templates was controlled for by parallel DMD-specific PCR amplifications. Ψ, cis-acting packaging elements; LTR, retroviral long terminal repeats; ITR, adenoviral inverted terminal repeats; half-arrows, primers drawn in relation to their respectively templates.

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Holkers, M., Maggio, I., Henriques, S. et al. Adenoviral vector DNA for accurate genome editing with engineered nucleases. Nat Methods 11, 1051–1057 (2014). https://doi.org/10.1038/nmeth.3075

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