Chapter 5 —— 118 —— joining processes (Miller et al. 2004; Hanlon et al. 2019; Ferrari et al. 2022; Li et al. 2024). Moreover, recent studies have demonstrated the pervasiveness of additional genomic DNA byproducts consisting of heterogeneous AAV vector-derived fragments and concatemeric species at off-target and on-target sites (Hanlon et al. 2019; Ferrari et al. 2022; Suchy et al. 2024; Li et al. 2024). The former AAV fragment intermediates, known to be packaged in vector particles (McColl-Carboni et al. 2024), typically contain transcriptionallycompetent ITR elements (Flotte et al. 1992; Haberman et al. 2000), raising transcriptome deregulation and insertional oncogenesis concerns (Ferrari et al. 2022; Bazick et al. 2024). Finally, when combined with programmable nucleases, AAV vector genomes exacerbate P53 build-up and ensuing DDR activation that impairs cell viability in a strict vector dose-dependent manner (Schiroli et al. 2019; Allen et al. 2022). We hypothesized that marker-free co-selection systems can be co-opted for addressing the aforementioned AAV-based genome editing shortcomings. These systems require neither chromosomal integration of exogenous selectable markers nor cell isolation reagents and equipment (e.g., FACS and MACS) (Mikkelsen and Bak, 2023). For this study, we selected a strategy based on ouabain, a highly potent and specific inhibitor of the ubiquitously expressed Na+/K+ ATPase pump (Agudelo et al. 2017). In contrast to other marker-free co-selection systems, such as that based on the potent inhibitor of mammalian protein synthesis diphtheria toxin protein, ouabain-dependent systems require a cheap small-molecule drug that has been used for congestive heart failure (Wu et al. 2015). Moreover, distinct ATP1A1 polymorphisms confer resistance to a broad range of ouabain concentrations that can be exploited for sequential installation of distinct genomic edits (Levesque et al. 2022), including those underpinning regulatory systems, complex gene circuits and other synthetic biology devices. For instance, the herein tested polymorphisms T804N, located in the third extracellular loop, and Q118R/N129D located in the first extracellular loop of Na+/K+ ATPase, create variants resistant to ouabain inhibition at 10 μM and over 1000 μM,
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