Generation of KI monitoring cell lines
The promoter-trap based KI monitoring CHO-K1 cell line harboring promoter-less EGFP expression cassette was generated in the previous study (Lee et al., 2018). Briefly, targeted integrants expressing GFP in the non-coding region of the CHO-K1 genome were transfected with two sgRNA expression vectors targeting entire CMV promoter regions, upstream of EGFP coding sequences, and then targeted clonal cells were isolated (Lee et al., 2018). The double KI monitoring CHO-K1 cell line was generated from the aforementioned single KI monitoring cell line. The single KI monitoring cell line was co-transfected with Cas9/sgRNA expression vector targeting COSMC locus and donor plasmid containing puromycin and promoter-less TagRFP657 expression cassette as described in Figure S4A. After two weeks of puromycin selection, a limiting dilution was conducted for single cell cloning. The selected clones were screened by out-out and 5’/3’ Junction PCR (Figure S4B), and maintained in culture media with puromycin (3 µg/ml) and G418 (500 µg/ml). The adherent cell lines were adapted to serum free suspension culture. Cells were seeded at 5.0×105 cells/ml in 6-well clear flat bottom not treated cell culture plate (Falcon) with suspension culture media, Power CHO medium (Lonza) supplemented with 8 mM L-Glutamine (Hyclone). The fetal bovine serum (FBS, Hyclone) gradually decreased from 3% to 0% until viable cell density reached up to 1.0 – 1.5 ×106 cells/ml.