Efficient Cell Line Generation using MIN-Tagging and Bxb1

To produce MIN-tagged cell lines, 5 × 10⁵ cells were dissociated and seeded in 0.2% gelatin (Sigma-Aldrich) coated p35 plates. After 3 h, cells were transfected with 2 μg of the MIN-tag donor/homology ssDNA oligo or PCR product, 0.5 μg gRNA construct, 0.5 μg surrogate reporter construct and 1 μg Cas9 using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. For Bxb1-mediated recombination of attB constructs, 5 × 10⁵ cells were transfected with 1 μg pCAG-NLS-HA-Bxb1 expression plasmid ((26 (link)) addgene 51271), 1 μg of the respective attB construct and 0.5 μg Bxb1 surrogate reporter. For both MIN-Tagging and Bxb1-mediated recombination, cells were dissociated, resuspended in ESC medium 48 h post transfection and then analyzed and sorted with a FACS Aria II (Becton Dickinson). For MIN-tagging, enrichment of cells with RGEN activity was accomplished by single-cell sorting GFP and mCherry positive cells into 96-well plates (Falcon) containing 150 μl of ESC medium. For Bxb1-mediated recombination, cells with Bxb1 activity were enriched for by single-cell sorting GFP positive cells into 96-well plates. Alternatively for Bxb1-mediated integration using antibiotic selection, cells were replated into p150 plates with ESC medium containing G418 (0.5 mg/ml, AppliChem) and puromycin (1 μg/ml, AppliChem) 48 h post transfection.