Chapter 4 —— 43 —— Cas9 and single or multiple gRNAs. Conversely, recombinant AAV genomes serve as proficient substrates for HR, including in difficult-to-transfect stem cell populations [15], and for NHEJ-mediated HITI in vitro and in vivo [5]. Possibly, the peculiar structure of AAV vector genomes featuring secondarystructured ITRs flanking single-stranded DNA contributes to their recombinogenic character [16,17]. Hence, here, inspired by the complementary attributes of these viral vector platforms, we sought to investigate genome editing strategies based on allocating AdV and AAV systems for the delivery of, respectively, CRISPR-Cas9 nucleases and distinct types of donor DNA templates. In particular, second- and third-generation AdVs provided for Cas9 nucleases, whilst AAVs served as sources of HR and HMEJ donor substrates presented as single-stranded or double-stranded DNA molecules. In addition, to further study the role of AAV donor DNA structures on the efficiency and accuracy of genome editing, standard and self-complementary (sc) AAV vectors were assembled to present in target cell nuclei donor templates in single-stranded and double-stranded DNA formats, respectively. Similarly to AAVs, third-generation AdVs, hereafter named high-capacity AdV particles (AdVPs), consist of viral gene-free recombinant DNA packaged in non-enveloped protein capsids [18,19]. In contrast to highcapacity AdVPs, first- and second-generation AdVs lack only a limited number of coding regions, e.g. early (E) region 1 (E1) alone or together with E2A or E4. These viral gene containing AdV vector genotypes can further possess deletions in immunomodulatory E3 sequences whose functions are dispensable during vector amplification in complementing cell lines [10,11]. Co-transduction experiments using early-generation AdVs encoding CRISPR-Cas9 complexes and matched AAV donors tailored for HR or HMEJ yielded limited gene targeting due to strict dose dependent cytotoxicity likely associated with ‘leaky’ AdV gene expression in target cells. Co-transduction experiments using fully viral gene-deleted AdVPs and the aforementioned AAV donors led instead to robust gene targeting without noticeable
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