Cloning, transfection, and lentiviral transduction

For in vivo CRISPR, two sgRNA-targeting mouse Mga (sgMga#1 and sgMga#3) were designed and cloned into the lentiCRISPRv2cre vector (Walter et al., 2017). See Key resources table for sequences. Virus was generated by the Fred Hutch Cooperative Center for Excellence in Haematology Viral Vector core and titered using qPCR. For in vitro studies in human lung cancer lines, a guide RNA against MGA was cloned into the lentiCRISPRv2 puro vector (kind gift from Feng Zhang). Viral supernatant was generated via transfection of 293FT or 293TN cells with lentiCRISPR v2 constructs and packaging vectors pPAX2 and pVSV-G using lipofectamine 2000 (Invitrogen). Supernatant was cleared of cellular debris using a 0.45 µM filter, and target cells were transduced with viral supernatant containing 4 µg/ml polybrene final concentration. Cell were grown in 10 µg/ml puromycin 3 days post-transduction. For addback of MGA or MGAΔDUF to mouse KP lines, cells were transfected on chamber slides with pCDH-MGA-FLAG or pCDH-MGAΔDUF-FLAG using lipofectamine 2000 (Invitrogen). Cells were harvested 3 days post-transfection and fixed for immunofluorescent staining. The cloning strategy was also used for cloning a sgRNA into the lentiCRISPRv2 construct with guide RNAs against human PCGF6, human L3MBTL2, mouse Pcgf6, mouse L3mbtl2, and mouse Myc. See Key resources table for sequences.