Chapter 5 —— 107 —— Recently, we introduced and characterized a dual viral vector genome-editing system based on the delivery of CRISPR-Cas9 nucleases and donor DNA templates via high-capacity adenovector particles (AdVPs) and AAV vectors, respectively (Li et al., 2024). Earlier experiments from our laboratory and those of others have shown that, contrary to linear free-ended DNA, capped double-stranded DNA, including adenovector genomes, are refractory to endjoining processes underpinning off-target and random chromosomal DNA insertions (Holkers et al. 2014; Medert et al. 2023). Moreover, besides their vast packaging capacity (i.e., up to 36 kb), viral gene-free AdVPs display a remarkably lower cytotoxicity profile when compared to that of their viral gene-containing, earlier-generation, counterparts (Li et al. 2024; Brescia et al. 2020; Tasca et al. 2020; Ricobaraza et al. 2020). Hence, to expand and streamline the testing of selector AAV vectors in established cell lines as well as in difficult-to-transfect primary cells, we used this dual viral vector platform in HeLa cells and human mesenchymal stem cells (hMSCs). Initial co-transduction experiments in HeLa cells using AdVP.C9KARA, a vector encoding the high-specificity nuclease SpCas9KARA (Figure 2A) (Wang et al. 2021), and different amounts of AAV-HRS1.A1, led to a clear dose-dependent increase in productive AAV transduction as assessed by EGFP-directed flow cytometry at 3 days post-transduction (Figure 2B). The significant AdVP.C9KARA-dependent enhancement on productive AAV-HRS1.A1 transduction (Figure 2B) is mostly caused by the higher transgene expression levels resulting from the buildup of chromosomally targeted templates over non-integrated episomes known to be prone to cellular restriction factors (Li et al., 2024; Dever et al. 2016). Moreover, earlier experiments have also established a causal relationship between CRISPR-Cas9-induced DSBs and productive AAV transduction (Li et al. 2024). Most importantly, after a 20day sub-culturing period in the presence and absence of ouabain, EGFPdirected flow cytometry revealed significantly higher frequencies of stably transduced cells in the presence of ouabain (Figure 2C). Of notice, the lowest and highest stably transduced cell fold-enrichment factors were associated
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