Chapter 6 —— 157 —— streamlining and expanding selector AAV vector testing into hard-totransfect primary cells. As covered in Chapter 2 and Chapter 3, there is a growing realization that, especially in DNA damage sensitive stem cells, programmable nucleaseinduced DSBs are detrimental to target loci stability [22,23], and cell viability [7,19]. Significantly, when compared with Cas9 nucleases, Cas9D10A nickases are notoriously less mutagenic at both on-target and off-target genomic sequences as determined by reporter and unbiased genome-wide highthroughput assays, respectively [24,25]. Cas9D10A nickases further display greatly reduced P53-dependent DDR activation when tested side-by-side with their Cas9 nuclease counterparts [9,26]. In this context, transduction experiments described in Chapter 5 have shown that combining a particular selector AAV design, dubbed in-linkage selector AAV, with Cas9 or Cas9D10A exposure leads to a significant increase in the frequencies of genetargeted cells. The ouabain-dependent enrichment factor for gene-targeted cells was circa 7.5-fold higher when using Cas9D10A nickase delivery. Importantly, combining in-linkage selector AAV transduction with Cas9 nuclease or Cas9D10A nickase delivery led to the thorough elimination of donor DNA inserted at off-target genomic positions from genome-edited cell populations. Therefore, these proof-of-principle experiments indicating that selector AAV-based genome editing is transportable to protocols involving Cas9D10A-induced HDR are relevant for furthering the refinement and application of DSB-independent cell engineering strategies. In conclusion, through the investigation and integration of distinct viral vectors and RNA-programmable Cas9 proteins, the work presented in this thesis provides new insights and toolboxes for advancing genome editing through the improvement of performance aspects related to its efficiency, specificity and fidelity.
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