Chapter 5 —— 124 —— 14040-091) containing 0.001% Poloxamer 188 (Sigma-Aldrich; Cat. No.: P5556). The purified batches of AAV-HRS1.A1, AAV-HRLMN.A1, and AAVHRA1.IN17 were stored at -80℃ and their transducing unit (TU) titers were determined by end-point titrations on HeLa cells using flow cytometry or qPCR assays as readouts. For AAV-HRS1.A1 and AAV-HRA1.IN17 titrations, HeLa cells were seeded at a density of 5×104 cells per well of 24-well plates (Greiner Bio-One) and approximately 18 hours later, they were incubated with 3-fold serial dilutions of each vector batch. The frequencies of transduced cells were determined at 3 days post-transduction by EGFP- or mScarlet-directed flow cytometry with the functional AAV vector titers corresponding to TU per ml being determined as the percentage of transduced cells × number of cells seeded × dilution factor x 1000 μl-1. The transducing titers of AAV-HRLMN.A1, expressed in GC per ml, were determined via a qPCR assay based on the iQ SYBR Green Supermix (Bio-Rad, cat. No. L010171C) and the mScarlet-I-specific primers 5’-CTACCTGGCGGACTT CAAGA-3’ and 5’-ACGGTGTAGTCCTCGTTGTG-3’. In brief, HeLa cells were seeded at 8.5 × 104 cells per well of 24-well plates (Greinder Bio-One) and, the next day, they were exposed to seven 3-fold serial dilutions of purified vector stock. Next, qPCR analysis was performed on genomic DNA isolated via the DNeasy Blood & Tissue kit at 24 hours post-transduction. In parallel, eight serial 10-fold dilutions of linearized parental AAV vector DNA containing 1×107 GC ml-1 served to setup a qPCR standard curve. Data analysis was done with the Bio-Rad CFX Manager 3.1 software (Bio-Rad Laboratories) and the titer was determined on the basis of the AAV vector DNA and plasmid Ct value standard curve. Testing selector AAV genome editing at AAVS1 and ATP1A1 HeLa cells were seeded at the density of 5 × 104 cells per well of 24-well plates. The next day, the cells were exposed or not to complexes formed by incubating 150 mM NaCl solutions containing 2.19 μl of PEI (1 mg ml-1) and plasmid mixtures consisting of 420 ng of AV62_pU.CAG.Cas9.rBGpA or 420 ng AB66_pU.CAG.dCas9.rBGpA as negative control. To control for
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