AAV genome quantification by droplet digital PCR
Genome-containing viral particles were titrated via ddPCR [57]. Viral DNA from samples was extracted using a High Pure Viral Nucleic Acid Extraction kit (Roche Diagnostics, Switzerland). The following primers targeting the transgene were used: EGFP, 5’-CTGCTGCCCGACAACCAC-3’ (forward) and 5’-TCACGAACTCCAGCAGGAC-3’ (reverse); SaCas9, 5’-GGCCAGATTCAGGATGTGCT-3’ (forward) and 5’- CATCATCCCCAGAAGCGTGT-3’ (reverse). The primers were purchased from Integrated DNA Technologies (USA). The thermocycling temperature programming for EGFP was: preincubation at 95°C/15 min for denaturation. 40 cycles of 94°C/30 s, 53°C/30 s and 72°C/1 min, and final extension at 72°C for 5 min. For SaCas9 was: preincubation at 95°C/15 min for denaturation. 40 cycles of 94°C/30 s, 60°C/1 min and 72°C/30 s, and final extension at 72°C for 5 min. The plates were scanned on a QX100 droplet reader (Bio-Rad, USA), and the analysis was carried out with QuantaSoft software (Bio-Rad, USA). Quantification was performed as previously described [58]. Cell-specific viral yield (CSVY) was determined by dividing the titer by the cell density at the time of transfection.