Chapter 5 —— 113 —— subjected to clonal analysis for characterizing genome-modifying events at the single-cell level (Figure 4C). At 3 days post-transduction, AAVHRA1.IN17 donor delivery into virtually all HeLa cells was achieved (Figure 4D, left panel). At 20 days post-transduction, ouabain untreated cultures initially exposed to Cas9 and Cas9D10A had circa 60% and 7.4% of genomemodified cells, respectively (Figure 4D, right panel). This difference is consistent with single-strand DNA breaks (SSBs), or nicks, being generally weaker HR stimuli than DSBs (Chen et al. 2017), including when using AAV HR donors (Pavani et al. 2021). Despite this, nickase-based genome editing offers notable advantages that include a striking reduction in off-target effects and on-target allelic mutagenesis as, in contrast to DSBs, SSBs are typically not engaged by error-prone end joining repair pathways (Chen et al. 2017). Importantly, addition of ouabain to cultures transduced with AAV-HRA1.IN17 and exposed to Cas9 or Cas9D10A led to a significant increase in the frequencies of genome-modified cells, i.e., 1.6- and 12-fold, respectively (Figure 4D, right panel). Moreover, as previously observed when using the bipartite donor AAV-HRS1.A1 (Figure 1D and Supplementary Figure S1), HeLa cell cultures exposed exclusively to AAV-HRA1.IN17 contained a low, yet clearly measurable, proportion of stably transduced cells (3.5%). The selective abolishment of this cell fraction in the presence of ouabain (Figure 4D, right panel), indicates that it results from the random chromosomal insertion of vector DNA. In line with this, data from the subsequent junction PCR screening of 160 arbitrarily isolated mScarlet-positive clones (80 expanded from cultures exposed to Cas9 and 80 expanded from cultures exposed to Cas9D10A), was consistent with the absence of randomly inserted donor DNA in cells grown in the presence of ouabain (Figure 4E and Supplementary Figure S4). Equally reminiscent of the results obtained with the bipartite donor AAV-HRS1.A1 (Figure 1F and Supplementary Figure S2), next to off-target DNA insertion purging, combining ouabain incubation with in-linkage donor AAV-HRA1.IN17 delivery also yielded a substantial reduction
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