Viral production and multiplicity of infection (MOI) estimation

To optimize conditions for viral transduction, virus was produced in parallel in 10 cm dish- and 96-well format. For 10 cm dish-format, lentiviral particles were produced by transfecting HEK293T packaging cells at 80% confluence with 5 μg of the sgRNA lentiviral construct, 3.75 μg of psPax2 packaging plasmid, 1.25 μg of pMD2G envelope plasmid and 1 μg pAdVantage (Promega E1711) at a ratio of 3:1 DNA to FugeneHD (Promega E2311). For 96-well format, the number of cells and the amount of plasmid DNA was reduced 40 times. Approximately 24 h and 48 h after transfection, the supernatant containing viral particles was recovered, filtered through a 0.45 μm filter (Millipore SLHV033RS or MSHVS4510), pooled together, and frozen at -80^oC. In order to determine the multiplicity of infection (MOI) of the virus, 1 × 10⁵ fibroblasts were seeded per well of a 6-well plate and infected with serial dilutions of virus generated in 10 cm dish-format, starting from 1:1. Following selection with 4 μg/mL blasticidin, the number of viable cells/well were measured and compared to a non-infected control to calculate the percentage of infected cells. The formula P = 1-e^−m, where P is percentage of infected cells and m represents MOI, was used to determine the viral concentration yielding the desired MOI in 96-well format (typically MOI = 1).