Chapter 6 —— 155 —— introduced, characterized, and tested for HDR-mediated gene KI purposes. In this dual viral vector genome-editing platform, RGN constructs are packaged in AdV capsids; whilst donor DNA templates are placed in AAV capsids. Next to genome-editing tool delivery considerations linked to the ultimate efficiency of gene KI procedures, other parameters to consider regard the specificity and precision of donor DNA insertion. The specificity results from detecting donor DNA sequences at the target site; whilst the precision can be assessed by establishing the precedence of accurate HDR-dependent DNA insertions over imprecise NHEJ-derived events. In this regard, knowledge concerning the relative contributions of distinct AAV donor designs (i.e., conventional versus ‘double-cut’) and structures (i.e., single-stranded versus double-stranded) to the genome-editing specificity and precision parameters, is scant. Hence, besides achieving remarkably high gene KI efficiencies in transformed, non-transformed and primary cells, the dual viral vector platform studied in Chapter 4 served as a probe to study the relationship between the aforementioned AAV DNA arrangements and the specificity and precision of gene KI and tagging procedures. This research disclosed that combining single-stranded AAV delivery of HDR donors with high-capacity AdV transfer of RGN constructs leads to precise genome editing in large fractions of target-cell populations. And, it revealed that RGN-induced chromosomal DNA breaks promote productive AAV vector transduction as scored by transgene expression presumably owing to fostering singlestranded to transcriptionally-competent double-stranded DNA conversion. Finally, in Chapter 4, it is further disclosed the critical importance of using high-fidelity RGNs for minimizing off-target donor AAV insertions in the form of defective vector genomes known to be packaged in vector particles [14,15]. The aforementioned recombinogenic character of AAV vector genomes, that makes them proficient HDR substrates, also contributes to their promiscuous “capture” at on-target and off-target or random chromosomal DNA breaks
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