Chapter 5 —— 102 —— The performance of marker-free co-selection systems in the context of viral vector delivery is presently unknown. Moreover, their utility for purging genome-edited cell populations from off-target as well as imprecise on-target chromosomal insertions is also underexplored. To fill these knowledge gaps, in this study, we set out to investigate AAV donor constructs harbouring marker-free co-selection components (selector AAV vectors) allowing for ouabain-dependent enrichment for genome-edited cells (Agudelo et al. 2017). We demonstrate that combining selector AAV vectors with ouabain treatments, next to enriching for genome-edited cell populations, achieves concomitant elimination of imprecise on-target edits and off-target and/or random donor DNA insertions from said populations. Interestingly, selector AAV vector titration experiments revealed that the highest fold-enrichment factors of genome-edited cell fractions are associated with the lowest vector input amounts which are expected to be beneficial for alleviating AAV vector production costs, off-target donor insertions and P53-dependent DDR activation. Results To start investigating AAV-based genome editing involving marker-free ouabain co-selection (Figure 1A), we assembled the selector vector AAVHRS1.A1. This vector contains HR donor templates and matched gRNA units designed for CRISPR-Cas9-induced transgene insertion at the human AAVS1 safer harbour locus (19q13.4-qter); and generation of ATP1A1 alleles with the Q118R and N129D (RD) polymorphisms conferring resistance to ouabain (Figure 1B). The AAVS1- and ATP1A1-specific gRNAs are complementary to intronic sequences to mitigate NHEJ-mediated mutagenesis of target alleles and both HR templates and cognate gRNA units are packaged in single AAV particles to guarantee their co-delivery into individual cells. Amongst the increasing range of genome editing strategies, gene knock-in into genomic safe harbour loci remains a particularly flexible approach as it permits to, for instance, correct in a predictable and safe manner the phenotype(s) of recessive disorders regardless of their causative mutations (Pavani and
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