Chapter 5 —— 115 —— To test the performance of the selector AAV in-linkage design in terms of its robustness for ouabain-dependent selection of gene targeted cells (Figure 5A), hMSCs and HeLa cells were co-transduced with AdVP.C9KARA and AAV-HRA1.IN17 (Figure 5B and 5C, respectively). As aforementioned, the observed AdVP.C9KARA-dependent enhancement on productive AAV transduction (Figure 5B and 5C) is primarily caused by the higher transgene expression resulting from the accumulation of chromosomally targeted exogenous DNA, known to be more refractory to cellular restriction factors than non-integrated episomal DNA (Dever et al. 2016; Li et al., 2024). Critically, after a sub-culturing period in the presence and absence of ouabain, mScarlet-directed flow cytometry disclosed remarkably strong ouabaindependent positive selection that consistently yielded over 90% of gene targeting frequencies independently of selector AAV vector doses and transduced cell type (Figure 5D and 5E). characterization of genome editing outcomes. The frequencies of the different types of genome-modifying events detected in cell clones randomly isolated from HeLa cell populations stably transduced with AAV-HRA1.IN17 donor DNA and expanded in the presence and absence of ouabain are plotted. Gene targeting events derived from precise HR at the telomeric and centromeric side of the target sequence are marked in cyan. Gene targeting events involving partial HR or no HR are labelled in orange and yellow, respectively. Off-target donor DNA insertion events are coloured in red (Supplementary Figure S4).
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