Chapter 5 —— 112 —— Hence, we sought to investigate whether a more strict enrichment for genomeedited cells with sustained purging of random and/or off-target insertions is achievable by using selector AAV donors with transgenic DNA juxtaposed to a selectable polymorphism (Figure 4A). To this end, the vector AAVHRA1.IN17 was assembled (Figure 4B). This vector contains a gRNA unit for directing cleavage at intron 17 of ATP1A1 and a matched donor DNA template designed for (i) mScarlet-I transgene knock-in at this intron; and (ii) installing the ouabain-selectable polymorphism T480N at the contiguous exon 17 (Agudelo et al. 2017). We started testing this selector AAV-HRA1.IN17 vector with in-linkage transgene and T840N by transducing HeLa cells transfected with plasmids expressing Cas9 nuclease or Cas9D10A nickase proteins. After sub-culturing in the presence or absence of ouabain, stably transduced cells were quantified by mScarlet-I-directed flow cytometry and AdVP.C9KARA was monitored through mScarlet-directed flow cytometry and genotyping assays based on junction PCR and RFLP assays (not shown). (B) Quantification of selector AAV gene tagging with and without ouabain. Gene tagging was determined in HeLa cell populations initially exposed to the indicated vector doses via mScarlet-directed flow cytometry at 14 days post-transduction. Mock-transduced cells and cells transduced exclusively with AAV-HRS1.A1 at 4,000 GC cell-1, served as negative controls. The data are shown as mean ± SD of three biological replicates. Significant differences amongst the marked datasets were calculated by Student’s t-tests; **P<0.05. (C) Genotyping of LMNA and ATP1A1 in co-transduced cell populations. LMNA and ATP1A1 gene editing in HeLa cells co-transduced with the indicated vectors and incubated with or without ouabain was assessed through junction PCR and RFLP assays (left and right panels, respectively). Mock-transduced cells served negative controls. (D) Characterization of LMNA protein tagging. LMNA tagging and nuclear localization was monitored by combining direct fluorescence microscopy for mScarlet expression and nuclei labelling using the DNA dye Hoechst 33342. (E) Assessing ouabain-dependent selection of ATP1A1 edited cells. HeLa cells co-transduced with AAV-HRLMN.A1 and AdVP.C9KARA and then cultured with or without ouabain were either sorted or not sorted to isolated LMNA-tagged positive and negative cells. ATP1A1 editing in each cell population was probed through RFLP assays. Mock-transduced cells and cells exposed only to AAV-HRLMN.A1 at 4,000 GC cel l-1, served as negative controls.
RkJQdWJsaXNoZXIy MTk4NDMw