Chapter 5 —— 114 —— of imprecise non-homologous recombination (Figure 4E and Supplementary Figure S4). Figure 4. Testing and characterizing selector AAV genome editing based on gene knock-in and marker-free selection linkage. (A) Gene targeting with off-target insertions purging via gene knock-in and marker-free selection linkage. HR donors designed for concomitant knock-in of transgenes and dominant ouabain-resistance polymorphisms are postulated to achieve a strict enrichment for gene targeted cells with the simultaneous thorough eradication of random and/or off-target donor DNA insertions. (B) Selector AAV donor with in-linkage transgene and dominant polymorphism. The selector AAV-HRA1.IN17 vector harbours a gRNA unit specific for intron 17 of ATP1A1 and a matched HR donor whose region homologous to ATP1A1 encodes the ouabainresistance polymorphism T804N. LHA and RHA, “left” and “right” homology arms, respectively. (C) Schematics of the experimental setup. HeLa cells transfected with Cas9 nuclease or Cas9D10A nickase constructs are transduced with AAV-HRA1.IN17 and cultured in the presence or absence of ouabain. The frequencies of genome-modified cells and the characterization of genome editing events are subsequently assessed trough mScarletdirected flow cytometry and clonal screens using junction PCR analysis at ATP1A1, respectively. (D) Quantification of selector AAV donor delivery and DNA editing. HeLa cells subjected to Cas9 and Cas9D10A plasmid transfections were transduced with AAVHRA1.IN17 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 determined gene targeting frequencies (right graph). The crosses denote full cell death in ouabain-treated cultures exposed exclusively to AAV-HRA1.IN17. (E) Cumulative
RkJQdWJsaXNoZXIy MTk4NDMw