Genome Editing in Diverse Cell Lines

HeLa, DE4, HEK293AC2, mouse MC57 cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS and 1% L-glutamine, and K562 cells were cultured in Iscove's modified Dulbecco's medium, 10% FBS and 1% L-glutamine. CHO cells were cultured in ex-Cell-CD media (Sigma Aldrich, USA) with 2% L-glutamine. Cells were nucleofected using solution kits T and V (K562) (Lonza, USA) and a Amaxa^® Cell Line Nucleofector^® device as previously described (15 ,16 ) using protocols provided by Lonza (http://www.lonzabio.com/resources/product-instructions/protocols/). In brief, 1 × 10⁶ cells were transfected with 2 μg of ZFN or TALEN plasmid pairs or 2 μg of Dual-GALNT6-ZFN. For CRISPR/Cas9 CHO Cosmc targeting, 2 μg Cas9 and gRNA plasmids were nucleofected, and for pCMV-Cas9-GFP expressing KRAS gRNA 2 μg plasmid was used. Cells were exposed to a cold shock 30°C for 2 days post-transfection, and incubated one day at 37°C after which DNA of the cell pool was prepared using Nucleospin kit as recommended by the supplier (Machery-Nagel, USA).
For consecutive targeting of the KRAS locus, K562 cells were subjected to fluorescence-activated cell sorting (FACS) 3 days after nucleofection for isolation of the 2% most highly GFP fluorescent cells that were then cultured for about 1 week. Thereafter, an aliquot of the cell pool was analysed by IDAA (first hit), whereas the rest of the cells were subjected to another two rounds of nucleofection and FACS to produce second and third hit pools, respectively, Furthermore, after the third hit, cells were also single-cell plated in 96-well plates and expanded to clonal cell lines.