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.