1. Co-transfection of CRISPR plasmids and HDR templates into HEK 293FT cells.
2. Linearize 1–2 μg of targeting vector if possible by cutting once at a restriction site in the vector backbone near one of the homology arms or at the distal end of either homology arm.
3. Run a small amount of the linearized plasmid alongside uncut plasmid on a 0.8–1% (wt/vol) agarose gel to check for successful linearization.
4. Purify the linearized plasmid with the PCR purification kit, and elute with EB buffer.
5. Preparation of cells for transfection. Culture HEK 293FT in T75 or T225 flasks. Plan ahead to have sufficient cells for the day of transfection.
6. Prewarming plates for transfection. Add 1 ml of warm D10 medium into each well of a 12-well plate. Place the plates in the incubator to keep the medium warm.
7. Preparing the co-transfection of the HDR targeting plasmid with the Cas9 plasmid.
8. For HDR applications, we recommend using 1 ug of Cas9 plasmid with sgRNA 1 ug of inearized targeting plasmid.
9. Dissociation of cells for transfection. Remove the medium and rinse the cells once gently with DPBS, taking care not to dislodge cells. Add 2 ml of TrypLE to a T75 flask and incubate it for 5 min at 37 °C, and then add 10 ml of warm D10 medium and triturate gently in a 50-ml Falcon tube.
10. Take a 10-μl aliquot from the cell suspension and dilute it into 90 μl of D10 medium for counting. Count the cells and calculate the number of cells and the volume of suspension needed for transfection.
11. Spin down the cells from Step 10 at 200g for 5 min at room temperature.
12. Prepare the transfection solution by mixing Lipofectamine 2000, Cas9 plasmid and inearized targeting plasmid.
13. Remove the medium completely from the pelleted cells from Step 11, and gently resuspend the cells in an appropriate volume of transfection solution for 4 hours.
14. Transfection solution is Gently pipetted and new media is added to cells.
15. Incubate the mixture for 24 h. At this point, transfection efficiency can be estimated from the fraction of fluorescent cells in the positive transfection control.
16. Slowly add 1 ml of warm D10 medium to each well without dislodging the cells. Puromycin selection can be applied at a concentration of 1−3 μg/ml for HEK 293FT cells (may vary depending on the cell line). Incubate the cells with puromycin for at least 72 h.
17. Single Cells are obtained by limiting-dilution assay and are cultured for 2 weeks.
18. Genomes of single cells are extracted for genotyping.
19. KO cell lines are selected by clonal sequencing validation.