Chapter 5 —— 106 —— can be enriched for when the latter locus acquires an edit(s) conferring a dominant resistance to a small-molecule drug. In this study, AAV-based genome editing of target and selectable ATP1A1 alleles is assessed in the presence and absence of ouabain, a highly potent and specific inhibitor of the essential Na+/K+ ATPase pump. (B) Schematics of selector AAV donor construct and selectable ATP1A1 site. The vector AAV-HRS1.A1 contains AAVS1 and ATP1A1 donor templates and cognate matched gRNA units that, in the presence of Cas9, trigger HR-mediated chromosomal insertion of a transgene and polymorphisms at the former and latter loci, respectively. The ATP1A1 polymorphisms Q118R and N129D (RD) confer resistance to ouabain. ATP1A1 editing can be probed via restriction fragment length polymorphism (RFLP) assays using BmgBI in untreated and ouabain-treated cell populations. (C) Schematics of the experimental setup. Cas9transfected HeLa cells are transduced with selector AAV-HRS1.A1 donor and, after subculturing in the presence or absence of ouabain, are subjected to EGFP-directed flow cytometry and to clonal screens using junction PCR analysis and RFLP assays at AAVS1 and ATP1A1, respectively. (D) Quantification of selector AAV donor delivery and DNA editing. HeLa cells subjected or not to Cas9 plasmid transfections were transduced with AAV-HS1.A1 at 1 TU cell-1. Donor delivery was assessed 3 days later by flow cytometry (left graph). After 20 days of sub-culturing in the presence and absence of ouabain, flow cytometry established AAV stable transduction frequencies (right graph). The cross indicates complete cell death in ouabain-treated cultures exposed exclusively to AAVHRS1.A1. (E) Characterization of genome editing outcomes. Left panel, representative clones yielding AAVS1 amplicons diagnostic for gene targeting involving precise HR events at the telomeric and centromeric side of the target sequence are marked by cyan arrowheads (jT and jC, respectively). Representative clones lacking AAVS1-specific insertions (off-target), containing HR-independent targeted insertions and with only one HR-derived junction between transgenic and AAVS1 sequences are marked by red, yellow, and orange arrowheads, respectively. Right panel, representative ATP1A1 amplicons resistant and susceptible to BmgBI digestion diagnostic for unedited and edited ATP1A1 alleles, respectively, are also depicted. (F) Cumulative 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 donor DNA and expanded in the presence or absence of ouabain are plotted (Supplementary Figure S2).
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