Chapter 5 —— 120 —— Selector AAV vectors with in-linkage selecting and targeting donor templates are, on the other hand, restricted to creating gene knock-ins at ATP1A1 alleles. Yet, ATP1A1 can in principle serve as a suitable transcription-favourable genomic landing pad to, for instance, overexpress proteins in producer cells, control cell behaviour with synthetic gene circuits, or complement genetic defects in autologous patient-derived cells. Moreover, albeit less versatile than bipartite donors, selector AAV vectors with in-linkage selecting and targeting HR templates yield engineered cell populations with substantially higher degrees of purity for gene targeted cells independently of vector doses and transduced cell types (range: 94.6%-99.4%). Indeed, the ability to generate such high frequencies of gene targeted cells using low AAV vector doses should allow creating genome engineered cells with minimal risks of harboring off-target and/or random chromosomal insertion of exogenous DNA (intact or otherwise). These favorable selector AAV performance features might permit streamlining cell engineering efforts via bypassing the need for time-consuming cell line isolation and screening and, in addition, expand said efforts to cell types refractory to single-cell isolation and expansion, such as most primary cells whose proliferation is restricted by their Hayflick limit and ensuing senescence. Equally of note, selector AAV vectors with in-linkage donor designs require single instead of dual CRISPRCas9 nucleases, therefore reducing genomic instability risks. There is nonetheless a growing realization that especially in DNA damage sensitive cells, like stem cells, programmable nuclease-induced DSBs can be detrimental to target locus stability (Frock et al. 2015; Kosicki et al. 2018) and cell viability (Chen et al. 2017; Ihry et al. 2018; Schiroli et al. 2019). Significantly, research from our laboratory has demonstrated that, when compared with Cas9 nucleases, Cas9D10A nickases are substantially less disruptive to on-target and off-target sequences (Wang et al. 2021; Chen et al. 2020) and present greatly dampened P53-dependent DDR activation levels (Wang et al. 2023). Hence, the herein provided proof-of-principle that selector AAV-based genome editing is transportable to protocols involving
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