Chapter 4 —— 44 —— cytotoxicity. In particular, combining single-stranded AAV delivery of HR donors with AdVP transfer of optimized high-specificity CRISPR-Cas9 nucleases yielded robust and precise gene targeting in human cells. We further disclose that productive AAV vector transduction directly correlates with the frequency of programmable nuclease-induced DSBs which may have a bearing on the ultimate performance and accuracy of AAV-based genome editing procedures. Finally, we demonstrate the importance of selecting highspecificity instead of regular Cas9 nucleases to avoid off-target chromosomal insertion of defective AAV DNA species in human cells. Results and discussion We started by investigating genome editing based on the delivery of donor DNA and CRISPR-Cas9 constructs in, respectively, AAV and secondgeneration AdVs lacking E1 and E2A. Gene knock-in into ‘safe harbor’ loci is, amongst the range of genome editing strategies, a particularly versatile approach in that it offers the prospect for correcting recessive disorders independently of their underlying mutations in a predictable manner. The predictability aspect stems from a minimization of insertional mutagenesis, transgene silencing and variegated expression, all valuable features associated with gene knock-ins at ‘safe harbor’ loci [38]. Hence, AAV vectors carrying HR or HMEJ donors were tailored for targeted knock-in of EGFP expression units at the commonly used human safe harbor locus AAVS1 at 19q13.4-qter (i.e. AAV-HRS1 or AAV-HMEJS1, respectively) (Figure 1A). Moreover, regardless of their genotypes, all adenovectors assembled for this study displayed adenovirus type-50 fibers. In contrast to prototypic type-5 fibers that recognize the coxsackievirus and adenovirus receptor (CAR), type50 fibers bind CD46 instead. As such, these tropism-modified vectors facilitate transduction of otherwise refractory cell types with potential or established therapeutic relevance, such as, CAR-negative human mesenchymal stem cells, muscle progenitor cells (myoblasts) and hematopoietic stem cells [39–41]. Relatedly, AAV donors consisted of recombinant AAV genomes based on type-2 packaged in AAV type-6 capsids
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