Chapter 5 —— 121 —— Cas9D10A-induced HR is relevant for further refining and applying markerfree co-selection approaches. In conclusion, in the present study, we integrate AAV HR donor delivery with a marker-free co-selection principle based on a commercially available and cheap small molecule, ouabain. We demonstrate that combining these selector AAV vectors with ouabain treatments, in addition to selecting for precisely edited cell populations, eradicates otherwise prevalent off-target and/or random AAV donor DNA insertions. Moreover, through selector AAV vector titration experiments, we report that the highest fold-enrichment levels for genome-edited cells is associated with the lowest vector inputs which is expected to be beneficial for mitigating AAV vector production costs and P53-dependent DDR activation processes known to limit AAV-based genome editing in DNA damage-sensitive cells. Selector AAV vectors are, therefore, expected to become useful in a broad array of basic and applied research contexts, such as for expediting cell engineering endeavours and generating well-defined populations of hard-to-transfect cell types including those that are refractory to clonal expansion but that hold nonetheless therapeutic relevance. Materials and Methods Cells The human cervix carcinoma HeLa cells (ATCC) were cultured in highglucose Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific; Cat. No.: 41966-029) containing 5% fetal bovine serum (FBS; Biowest; Cat. No.: S1810-500). The HeLa cells were kept at 37℃ in a humidified-air 10% CO2 atmosphere. The primary human mesenchymal stem cells (hMSCs) were isolated from bone marrow and cultured in Minimum Essential Medium α (MEM-α; Thermo Fisher Scientific; Cat. No.: 22561-021) supplemented with 10% FBS, 100 U ml-1 penicillin/streptomycin (Thermo Fisher Scientific; Cat. No.: 15140-122), 1× non-essential amino acids (NEAA; Thermo Fisher Scientific; Cat. No.: 11140-050), 1× GlutaMax supplement
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