Chapter 5 —— 101 —— particularly effective sources of donor DNA in a broad range of mammalian cell types (Epstein and Schaffer, 2017). Moreover, AAV vector genomes consisting of single-stranded DNA flanked by hairpin-forming inverted terminal repeats (ITRs), are prone to HR when harbouring sequences identical to those framing programmable nuclease target sites. Indeed, AAV HR donors yield high-efficiency gene targeting including in human cells with potential and established therapeutic relevance (Epstein and Schaffer, 2017). Unfortunately, their recombinogenic nature also contributes to off-target and imprecise on-target chromosomal donor DNA insertions involving nonhomologous recombination processes (Miller et al. 2004; Hanlon et al. 2019; Ferrari et al. 2022; Li et al. 2024). Other insidious byproducts include ontarget and off-target chromosomal insertion of, respectively, concatemeric structures and heterogeneous AAV DNA species that often, harbour ITR sequences (Hanlon et a. 2019; Ferrari et al. 2022; Li et al. 2024; Suchy et al. 2024). The latter events raise transcriptome deregulation and insertional oncogenesis risks due to the known transcriptional competency of ITR elements (Ferrari et al. 2022; Flotte et al. 1992; Haberman et al. 2000; Bazick et al. 2024). Equally of concern, AAV genomes, possibly due to mimicking DNA lesions or repair intermediates, can impair cell viability through P53dependent DNA damage response (DDR) activation whose consequences are particularly deleterious during stem-cell genomic engineering (Schiroli et al. 2019; Allen et al. 2022). Critically, imprecise and off-target byproducts as well as cytotoxic effects are strictly proportional to AAV vector amounts (Schiroli et al. 2019; Allen et al. 2022). Besides the efficiency, additional key parameters of genome editing procedures include their specificity and accuracy or fidelity (Maggio and Gonçalves, 2015). The former corresponds to the relative levels of on-target to off-target donor DNA insertions; the latter relates to the proportions between precise and imprecise on-target editing events.
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