Mpk5000

1 × 10⁵ PFF cells were harvested and resuspended in 10 μL electroporation buffer (MPK5000S, Invitrogen, Carlsbad, CA, USA) supplemented with hCas9, sgRNA, and reporter (molar ratio of Cas9:sgRNA:donor was 1:1:2, with a total of 2 μg). Reporter was digested with AhdI (R0584S, New England Biolabs) before transfection. The mixture was transfected into PFF cells using the Neon™ Transfection System (MPK5000, Invitrogen), with 1350 V/30 ms and 1 pulse. Subsequently, transfected cells were transferred to 12-well plates (3513, Corning, Corning, NY, USA), and EGFP expression was observed 48-h after transfection using fluorescence microscopy (Olympus, Shinjuku, Tokyo, Japan).
3 × 10⁵ PK15 cells were seeded into 12-well plates and reached 70–80% confluency at the time of transfection. 750 ng DNA (molar ratio of Cas9:sgRNA:reporter was 1:1:2) was transfected using 2.25 μL of GenJet™ In Vitro DNA Transfection Reagent (SL100489, SignaGen, Rockville, MD, USA). EGFP expression was observed 48 h post-transfection using fluorescence microscopy.

TracrRNA, crRNA, and Cas9 protein were purchased from IDT (Coralville, IA, USA). TracrRNA and crRNA were dissolved in Duplex Buffer (IDT, 200 µM each) and annealed in a thermal cycler at 95 °C for 10 min followed by − 1 °C/min stepdown cycles until 25 °C. Annealed RNA (100 µM) were then incubated with 3 µg/µL Cas9 protein at 37 °C for 20 min to form Cas9-RNP. A Neon Transfection System (MPK5000, ThermoFisher) was used for electroporation of plasmid and Cas9-RNP into ES cells. In brief, 1 × 10⁵ ES cells with 1 µg of targeting vector were electroporated with or without all-in-one CRISPR/Cas9 vector (0.5 µg) or Cas9-RNP (at a final concentration of 10 µM annealed RNA and 0.3 µg/µL Cas9 protein) in a 10 µL tip (MPK1096, Thermo). The Neon system used two pulses at 1200 V and 20 ms or a single pulse at 1400 V and 30 ms for all-in-one vector transfection or Cas9-RNP transfection, respectively. Electroporated ES cells were cultured in ESCM with or without MEFs.