All of the cell lines used in this study were tested for mycoplasma and were negative. RN2c cells were derived by retroviral transduction of a murine MLL-AF9/NrasG12D acute myeloid leukemia cell line (RN2)11 with MSCV-hCas9-PGK-Puro, followed by puromycin selection and serial dilution to derive single cell-derived clones. Clones were screened by anti-flag Western blotting for high levels of stable Cas9 expression. Multiple independent clones displayed a similar CRISPR editing efficiency as RN2c. RN2c were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. 38B9 cells were cultured in RPMI1640 supplemented with 10% FBS and 0.055 mM 2-mercaptoethanol. NIH3T3 cells were cultured in DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin. Ecotropic Plat-E cells and HEK293T cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Plat-E cells were used for retroviral delivery of hCas9 or RPA3 cDNA expression vectors, following standard procedures27 (link).
sgRNA competition assays were performed using the U6-sgRNA-EFS-GFP or the U6-sgRNA-EFS-mCherry plasmids, where indicated. These plasmids were used to generate lentivirus by transfecting HEK293T cells with sgRNA:pVSVg:psPAX2 plasmids in a 4:2:3 ratio using PEI reagent (Polysciences : #23966). Viral supernatants were collected between the 36 and 72 hour timepoints following transfection. All transfections and viral collections were performed in 96 well plates to allow the evaluation of large numbers of sgRNAs systematically in a one-by-one manner. For sgRNA/GFP competition assays, flow cytometry analysis was performed on 96 well plates of cells using a Guava Easycyte HT instrument (Millipore). Gating was performed on live cells using forward and side scatter, prior to measuring of GFP positivity.
For the Rpa3 sgRNA/cDNA rescue experiment, RN2c were first transduced with empty or RPA3 MigR1, followed by transduction with Rpa3 sgRNAs expressed using the U6-sgRNA-EFS-mCherry vector. Gating was performed on GFP+/mCherry+ cells to evaluate rescue.
sgRNA competition assays were performed using the U6-sgRNA-EFS-GFP or the U6-sgRNA-EFS-mCherry plasmids, where indicated. These plasmids were used to generate lentivirus by transfecting HEK293T cells with sgRNA:pVSVg:psPAX2 plasmids in a 4:2:3 ratio using PEI reagent (Polysciences : #23966). Viral supernatants were collected between the 36 and 72 hour timepoints following transfection. All transfections and viral collections were performed in 96 well plates to allow the evaluation of large numbers of sgRNAs systematically in a one-by-one manner. For sgRNA/GFP competition assays, flow cytometry analysis was performed on 96 well plates of cells using a Guava Easycyte HT instrument (Millipore). Gating was performed on live cells using forward and side scatter, prior to measuring of GFP positivity.
For the Rpa3 sgRNA/cDNA rescue experiment, RN2c were first transduced with empty or RPA3 MigR1, followed by transduction with Rpa3 sgRNAs expressed using the U6-sgRNA-EFS-mCherry vector. Gating was performed on GFP+/mCherry+ cells to evaluate rescue.
