HeLa cells were cultured in DMEM, 10% FBS, 1% Penicillin/Streptomycin and passaged with trypsin every 3–4 days. To generate the fluorescent reporter line, plasmid #208 was cloned. Successful integration into the AAVS1 loci generated neomycin resistance. Cells were selected with 0.6 mg ml−1 G418 for one week. Single cells were FACS sorted into a 96-well plate and expanded. Colonies were checked for correct integration by genotyping and a clone with inserts on both alleles was expanded and used. Targeting of Reporter HeLa: 50 000 cells were reverse-transfected with 1.5 µg of Cas9_2A_puro/guide plasmid + 1.5 µg of MC or plasmid complexed with Lipofectamine 3000. The next morning 1.5 µg ml−1 puromycin was added for 48 h. Cells were then FACS analysed. mCherry+ cells were single cell sorted into a 96-well plate and expanded for genotyping. For the HDR targeting experiment, the guide RNA targeting the Insert site was used together with the donor plasmid.
K-565, a leukaemia cell line, were kept in IMDM, 10% FBS, 1% penicillin/streptomycin and split every 3 days. For targeting, cells were nucleofected using the Lonza 4D strips. A total of 5 × 105 cells were resuspended in nucleofection buffer [56 ] with 1 µg Cas9/guide plasmid and 3 µg of minicircle and nucleofected using program FF-120. The following day puromycin (4 µg ml−1) was added for 48 h.
K-565, a leukaemia cell line, were kept in IMDM, 10% FBS, 1% penicillin/streptomycin and split every 3 days. For targeting, cells were nucleofected using the Lonza 4D strips. A total of 5 × 105 cells were resuspended in nucleofection buffer [56 ] with 1 µg Cas9/guide plasmid and 3 µg of minicircle and nucleofected using program FF-120. The following day puromycin (4 µg ml−1) was added for 48 h.