▲ CRITICAL The CRISPR-Cas system has been used in a number of mammalian cell lines. Conditions may vary for each cell line. Below we detail transfection conditions for HEK 293FT cells. Note that ssODN-mediated HDR transfections, performed with Amaxa SF cell line Nucleofector kit, are described in Steps 14–29. For hESC (HUES9) culturing and transfection, follow Steps 30–53. 6HEK 293FT maintenance. Cells are maintained according to the manufacturer's recommendations. Cells are cultured in D10 medium supplemented with 10% (vol/vol) FBS at 37 °C and 5% CO2.7To passage, remove the medium and rinse the cells once by gently adding DPBS to the side of the vessel, so as not to dislodge the cells. Add 2 ml of TrypLE to a T75 flask, and incubate the mixture for 5 min at 37 °C. Add 10 ml of warm D10 medium to inactivate the trypsin, and transfer the cells to a 50-ml Falcon tube. Dissociate the cells by pipetting them up and down gently, and then reseed them into new flasks as necessary.
