Chapter 5 —— 109 —— transduced cell populations. AAVS1 and ATP1A1 gene editing in HeLa cells co-transduced with the indicated vectors and incubated or not with ouabain was assessed through junction PCR and RFLP assays (left and right panels, respectively). Mock-transduced cells provided for negative controls. To test genome editing at an independent target locus, the selector AAVHRLMN.A1 donor was applied to HeLa cells and to primary hMSCs together with AdVP.C9KARA (Figure 3 and Supplementary Figure S3, respectively). The AAV-HRLMN.A1 vector contains the same selectable sequence of AAVHRS1.A1 together with a LMNA-specific gRNA unit and a matched HR template for tagging LMNA at its N-terminus with the live-cell reporter mScarlet-I (Figure 3A). Of note, LMNA mutations have been linked to, amongst others, Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. In contrast to the use of autonomous transgene expression units, gene tagging setups are contingent on precise gene knock-in to guarantee expression from endogenous cis-acting regulatory elements. As such, this setup directly traces and quantifies HR-mediated gene editing events. Cotransductions targeting LMNA alleles broadly recapitulated the results obtained through experiments targeting the AAVS1 locus (Figure 2). In particular, reporter-directed flow cytometry (Figure 3B and Supplementary Figure S3A), together with junction PCR and RFLP assays (Figure 3C, left and right panel, respectively) demonstrated an ouabain-dependent coselection for cells edited at target LMNA and ATP1A1 alleles. Again, the lowest and highest gene editing fold-enrichment factors resulted from applying the highest and lowest selector AAV doses, respectively (Figure 3B and Supplementary Figure S3A). The highest fold-enrichment factor (30fold) was, in fact, observed in hMSCs initially co-transduced with AdVP.C9KARA and AAV-HRLMN.A1 at 500 GC cell-1 (Supplementary Figure S3A). These results indicate the feasibility in generating high frequencies of genome-edited cell populations while using low amounts of AAV vectors known to trigger dose-dependent cytotoxic effects in cell types with
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