To construct MSCV expression plasmids expressing 3xFLAG-Sp-dCas9, the pMSCVneo, pMSCVhyg, and pMSCVpuro vectors (631461, Takara Bio, Kusatsu, Japan) were digested with BglII (1021A, Takara Bio) and XhoI (1094A, Takara Bio). After a blunting reaction, the plasmids were treated with bacterial alkaline phosphatase (E. coli C75) (2120A, Takara Bio). The cleaved vectors were purified by agarose gel electrophoresis and ligated with the coding sequence of 3xFLAG-Sp-dCas9, which was isolated from 3xFLAG-dCas9/pMXs-puro (Addgene (Watertown, MA, USA) #51240)  by digestion with PacI (R0547, New England Biolabs, Ipswich, MA, USA) and NotI (1166A, Takara Bio).
To construct gRNA-hIRF-1 #12/pSIR-hCD2, pSIR-hCD2 (Addgene #51143)  was digested with EcoRI (1040A, Takara Bio) and treated with bacterial alkaline phosphatase. The cleaved vector was purified by agarose gel electrophoresis and ligated with the gBlock targeting the interferon (IFN) regulatory factor (IRF)-1 gene promoter, which was isolated from gRNA-hIRF-1 #12 (Addgene #61079)  by digestion with EcoRI.
The Addgene plasmid # of the newly made constructs are as follows: 3xFLAG-Sp-dCas9/pMSCVneo: 134982; 3xFLAG-Sp-dCas9/pMSCVhyg: 134323; 3xFLAG-Sp-dCas9/pMSCVpuro: 134983; gRNA-hIRF-1 #12/pSIR-hCD2: 135392.
The 293T cell line was derived by transformation of human embryonic kidney (HEK) 293 cells with the SV40 large T antigen . HT1080 is a human fibrosarcoma cell line purchased from ATCC (Manassas, VA, USA) (CCL-121) . 293T, HT1080, and HT1080-derived cells were cultured in DMEM (FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal calf serum (FCS).
Transduction of retroviral plasmids
For transduction of retroviral plasmids expressing 3xFLAG-Sp-dCas9, 5.5 µg of each plasmid was transfected into 1 × 106 of 293T cells along with 5.5 µg of pPAM3  using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Two days after transfection, viral supernatant was harvested and used for infection of HT1080 cells. Infected cells were selected in culture media containing G418 (Nacalai Tesque, Kyoto, Japan) (0.8 mg/ml), hygromycin (Nacalai Tesque) (75 µg/ml), or puromycin (Nacalai Tesque) (0.75 µg/ml).
For transduction of retroviral plasmids expressing the gRNA, 5.5 µg of gRNA-hIRF-1 #12/pSIR-hCD2 was transfected into 1 × 106 of 293T cells along with 5.5 µg of pPAM3 using Lipofectamine 3000. Two days after transfection, viral supernatant was harvested and used for infection of HT1080 cells expressing 3xFLAG-dCas9. Cells expressing 3xFLAG-Sp-dCas9 and gRNA were sorted by MACS as described below.
Cytoplasmic extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Ten micrograms of cytoplasmic extract was subjected to immunoblot analysis with anti-FLAG M2 antibody (Ab) (F1804, Sigma-Aldrich, Saint Louis, MO, USA) as described previously .
MACS sorting and flow cytometry
MACS sorting of human CD2 (hCD2) (+) cells was performed using CD2 Microbeads (130-091-114, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany). To monitor the results of MACS sorting, cells were stained with phycoerythrin (PE)-conjugated anti-hCD2 Ab (347597, BD Life Sciences, San Jose, CA, USA) for 30 min at 4°C. Flow cytometry was performed on a FACSCalibur (BD Life Sciences), and data were analyzed using the FlowJo software (BD Life Sciences).
enChIP-real-time PCR was performed as previously described . The primers used in this study were reported previously .