Chapter 5 —— 132 —— Supplementary Figure S4. Characterization of ATP1A1 gene targeting resulting from selectable in-linkage AAV donor delivery. PCR genotyping of individual geneticallymodified cell clones generated via AAV-HRA1.IN17 transduction of HeLa cells transfected with a Cas9- or Cas9D10A-encoding plasmids. Prior to clone isolation, the HeLa cell populations were expanded either in the presence or absence of ouabain. Nuclease-free water (W) and DNA from unmodified HeLa cells (-) provided for negative controls; genomic DNA from sorted mScarlet-positive HeLa cells exposed to Cas9 or Cas9D10A and AAV-HRA1.IN17, served as positive controls (+). The ATP1A1 genotype screens involved junction PCR (jC and jT) analysis. Clones lacking ATP1A1 targeted insertions or with only one HR-derived junction between transgenic and ATP1A1 sequences are marked by red and orange arrowheads, respectively; whilst clones containing HR-independent donor DNA insertions are highlighted by yellow arrowheads. The remaining clones, modified through precise HR events involving the telomeric and centromeric side of the target sequence (jT and jC, respectively), are not highlighted. The cumulative datasets corresponding to these ATP1A1 genotype screens (n=160 clones representing independent genome-modifying events), are plotted in Figure 4E.
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