Chapter 5 —— 104 —— generated by AAV-HRS1.A1 delivery alone and together with Cas9, respectively (Supplementary Figure S1). As aforementioned, next to the efficiency, the specificity and accuracy of donor DNA integration are key parameters of genome editing procedures based on HR or otherwise. The specificity is defined by the presence of donor DNA at the target site whilst the accuracy results from generating seamless telomeric-sided and centromeric-sided junctions between exogenous and endogenous DNA (jT and jC, respectively). Therefore, to determine the specificity and accuracy of selector AAV-based genome editing in the presence and absence of ouabain, junction PCR screens were performed on isolated EGFP-positive cell clones, each of which representing individual genome-modifying events. Of notice, differently from splice acceptor genetrapping and protein-tagging constructs, clonal isolation based on transcriptionally autonomous transgenes, such as that in AAV-HRS1.A1, prevents the biased selection for on-target events. The screening of 160 randomly isolated clones (80 expanded with ouabain and 80 without), identified genome-modifying events corresponding to random or off-target donor DNA insertions (jT-/jC-), precise HR-mediated AAVS1 gene targeting (jT+/jC+), partial gene targeting (jT+/jC- or jT-/jC+), and non-homologous recombination, namely, containing differently-sized amplicon(s) diagnostic for imprecise DNA end-joining events (Figure 1E, left panel, red, cyan, orange and yellow arrowheads, respectively; and Supplementary Figure S2). Moreover, ouabain selection led to a remarkable increase in the number of ATP1A1 gene editing as traced through RFLP assays (Figure 1E, right panel and Supplementary Figure S2). Significantly, when compared with untreated cultures, cultures treated with ouabain contained substantially lower amounts of imprecisely edited cells (Figure 1F, top panel). Indeed, all the three categories representing imprecisely edited cells were smaller in ouabain-treated cultures (Figure 1F, top panel; and Supplementary Figure S2). The substantial enrichment for
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