CRISPR-Cas9 Knockout Protocol

1. HEK 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.

2. To 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.

3. Cells typically are passaged every 2–3 d at a split ratio of 1:4 or 1:8, never allowing cells to reach more than 70% confluency.

4. Antibiotics 16–24 h before transfection. Seed the cells at a density of 1.3 × 105 cells per well in a total volume of 500 μl. Do not plate more cells than the recommended density, as doing so may reduce transfection efficiency.

5. On the day of transfection, cells are optimal at 70–90% confluency. Cells can be transfected with Lipofectamine 2000. 2 ug of CRISPR plasmid is used.

6. Add Lipofectamine 2000 complex to the cells gently, as HEK 293FT cells can detach easily from the plate, which will result in a lower transfection efficiency.

7. Check cells after 24 h for transfection efficiency. The percentage of fluorescent cells in the transfection control (e.g., GFP) can be estimated by using a fluorescence microscope. Typically, more than 70% of cells are transfected.

8. Supplement the culture medium with an additional 500 μl of warm D10 medium.

9. Incubate the cells for a total of 48–72 h after transfection before passaging them for downstream applications or harvesting for indel analysis.

10. Media are discarded carefully and new media with Puro are added to cells gently. Cells are cultured for 3 days.

11. Single Cells are obtained by limiting-dilution assay and are cultured for 2 weeks.

12. Genomes of single cells are extracted and analyzed by sequnencing.

13. KO cell lines are selected by clonal sequencing validation.

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