Gecko library

Packaging and purification of lentivirus of sgRNA library were performed as previously described.¹⁷ Briefly, HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum (FBS). 4 μg of GeCKO library (#1000000048; Addgene), 2 μg of pV‐SVg (#8454; Addgene) and 6 μg of psPAX2 (#12260; Addgene) packing plasmids were cotransfected in a 10 cm dish using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's protocol. After transfection for 6 hours, the cell culture media was changed to flesh complete culture media. The media was collected after 48 hours and centrifuged at 1,000 g at 4°C for 20 minutes to remove the cell debris. The supernatant was filtered (0.45‐µm pore size) and concentrated by ultracentrifugation (Beckmann) at 24 000 rpm for 2 hours at 4°C. The virus preparation was finally resuspended with DMEM overnight at 4°C, divided into aliquots and stored at −80°C.

Lentivirus of sgRNA library was prepared following the protocol previously described (Cai et al., 2019 (link)). Briefly, HEK293T cells were divided into a 10 cm dish and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). GeCKO library (#1000000048, Addgene, Watertown, MA, United States), pV-SVg (#8454, Addgene) and psPAX2 (#12260, Addgene) packing plasmids were co-transfected at a mass ratio of 1:0.5:1.5 using Lipofectamine 3000 reagent (Invitrogen, San Diego, CA, United States) following the manufacturer’s protocol. 6 h after transfection, the cell culture media was replaced with fresh medium. The cell supernatant was collected and cell debris was removed by centrifugation at 3,000 rpm at 4°C for 30 min. After centrifugation, the supernatant was filtered with a 0.45-µm pore size filter and then ultracentrifuged at 24,000 rpm for 2 h at 4°C for concentration. Concentrated virus was resuspended with PBS overnight at 4°C, and stored at −80°C refrigerator.