GeCKO v2 lentiviral library production and transduction

We used the lentiCRISPRv2 human library designed by Shalem et al.⁵⁵ and obtained from Addgene. The sgRNA library was synthesized using array synthesis as previously described⁵⁵ and cloned as a pool into the lentiCRISPR transfer plasmid for virus production.

To produce the pooled lentiviral library, twelve T-225 flasks of HEK293T cells were seeded at ~40% confluency the day before transfection. Per flask 10 μg of pVSVg, and 15 μg of psPAX2 (Addgene) packaging plasmids and 20 μg of lentiCRISPR plasmid library were transfected using Lipofectamine 2000 and Plus reagent (Life Technologies), according to manufacturer’s instructions. After 6 hours the medium was changed, and after 60 hours the medium was collected and centrifuged at 3,000 rpm for 10 minutes at 4 °C to pellet cell debris. The supernatant was filtered through a 0.45 μm low protein-binding membrane (Millipore Steriflip HV/PVDF). To achieve a 300 times concentration of the GeCKO pooled library, the virus was ultracentrifuged (Sorvall) at 24,000 rpm for 2 hours at 4 °C and then resuspended overnight at 4 °C in D10 supplemented with 1% BSA. Aliquots were stored at −80°C.

Per condition 20 million MRC-5 cells were transduced at 75% confluency in 145 cm² dishes with concentrated lentivirus diluted in 18 ml of culture medium supplemented with 12 μg/mL polybrene (Sigma). The virus titer was determined to achieve a multiplicity of infection of <0.25. The next day, cells were re-seeded at 25% confluency in culture medium containing 2 μg/ml Puromycin. Cells were expanded for 1 week in puromycin-containing medium. Culture medium was refreshed every other day.