Lentivirus production and titration

For large-scale production, HEK293T cells were seeded in T225 flasks such that each flask would be ~80% confluent at the time of transfection. After overnight incubation, pCMV-VSV-G (Addgene #8454), psPAX2 (Addgene #12260), and pLGP-pgRNA transfer vectors were introduced into cells using PEI Max (Polysciences, Inc.) transfection. Lentivirus-containing media was harvested 48 hours later, filtered, and stored as 1 mL aliquots at −80°C until use. For small-scale production, HEK293T cells were seeded into individual wells of a 6-well plate and all reagents were proportionally scaled. To determine lentiviral titers, HeLa/iCas9 or PC9-Cas9 cells were seeded in individual wells of a 12-well plate in media supplemented with 8 μg/mL polybrene (EMD Millipore) and incubated at 37°C for 2 hours. Next, serial dilution of the lentivirus preparation was added to individual wells and incubated for 24 hours at 37°C. The next day, cells from individual wells of the 12-well plate were re-seeded into eight wells of a 96-well plate. Cells in four of these wells were grown in culture media supplemented with 1 μg/mL puromycin and the other four contained no puromycin. After all cells in the no-infection control wells were dead (typically 2–3 days), cell viability was quantified using a CellTiter-Glo (Promega) assay according to the manufacturer’s instructions. Multiplicity of infection was determined by calculating the ratio of cells in the puromycin treated compared to no puromycin treatment groups.