Chapter 5 —— 100 —— donor plasmid is co-transfected with a secondary donor construct or oligonucleotide designed for creating drug- or toxin-selectable dominant alleles with specific polymorphism(s) (Agudelo et al. 2017; Wiebking et al. 2020; Li et al. 2021). A marker-free co-selection strategy based on the acquisition of gain-of-function resistance to a highly potent and specific inhibitor of the sodium/potassium (Na+/K+) ATPase pump, namely the plantderived cardiotonic steroid ouabain, constitutes a particularly powerful coselection approach (Agudelo et al. 2017). The main attributes of this approach are two-fold. Firstly, it targets the essential and ubiquitously expressed ATP1A1 gene yielding, as a consequence, a robust and universal selectable phenotype; and, secondly, it is based on a commercially available and cheap small-molecule that, over decades, has been administered for congestive heart failure (Wu et al. 2015). Moreover, distinct ATP1A1 polymorphisms confer cellular resistance to a broad range of ouabain concentrations which can be exploited for reiterative implementation of distinct genomic edits within individual cells (Levesque et al. 2022). Plentiful physical and chemical transfection methods allow for introducing genome editing reagents into human cells including donor DNA substrates in the form of plasmids or synthetic oligonucleotides. However, achieving optimal transfection efficiencies without the build-up of cytotoxic effects is demanding as it often requires systematic cell type-specific protocol optimizations. Moreover, the ultimate performance of these optimized protocols, whose reagents are sometimes unknown due to proprietary reasons, typically depends on subtle experimental conditions, e.g., cell-cycle stage distributions during transfection. In contrast to transfections, viral vector transductions present higher reproducibility and can be directly applied to different cell types independently of their cell cycle statuses. Valuable viral vector delivery properties stem from the fine-tuned mechanisms evolved by their wild-type counterparts in delivering nuclei acids into the cytoplasm or nucleus of the host cell. In this regard, commonly used adeno-associated viral (AAV) vectors with regular, pseudotyped or engineered capsids are
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