Chapter 4 —— 71 —— precise HDR-derived events than their single-stranded or scAAV HR counterparts. Presumably, as for the other aforementioned types of vehicles, generation of free-ended double-stranded DNA from scAAV HMEJ substrates favors end-joining products at DSBs (targeted or otherwise). On the other hand, it will be valuable determining whether, similarly to ‘doublecut’ AAV HITI donors, AAV HMEJ donors also outperform AAV HR designs in non-dividing or post-mitotic cells. Ultimately, the selection of individual AAV donor substrates will depend on a judicious assessment of their performance taking into account the goal and specific settings in which they are meant to operate. Regardless, in the context of AAV-based genome editing procedures the use of high-specificity CRISPR-Cas9 complexes is critical to mitigate HDR-independent insertions of defective AAV genomic species at off-target chromosomal positions. In conclusion, the dual viral vector platform introduced herein permits investigating different genome-editing reagents and strategies and offers the prospect for the effective and accurate chromosomal edition of hard-totransfect cell types with scientific and therapeutic relevance. Applications may include establishing human disease-in-a-dish models and engineering cellular substrates for drug screens and, eventually, autologous cell therapies. Materials and methods Cells The human embryonic kidney 293T (HEK293T) cells (ATCC) and the human cervix carcinoma (HeLa) cells (ATCC) were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific; cat. no.: 41966–029) containing, respectively, 10% and 5% fetal bovine serum (FBS; Biowest; cat. no.: S1810-500). Both cell lines were cultured at 37℃ in a 10% CO2 atmosphere. The PEC3.30 packaging cell line used for the assembly of AdVPs [20], were cultured in high-glucose DMEM with 10% FBS, 10 mM MgCl2 and 0.4 μg ml -1 puromycin (Thermo Fisher Scientific; cat. no.: A11138-03) at 39℃ in a 10% CO2 atmosphere. The bone marrow-
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