Nucleofection: The sgRNAs targeting specific genes are designed according to a method provided online (http://crispr.mit.edu/). 2. The sgRNA oligos are annealed and ligated to the Cas9 plasmid that contains the mCherry reporter. Prepare Cas9 plasmid using endotoxin-free plasmid kit. 3. About 7 days before nucleofection, passage SSCs at a dilution of 1:3 or 1:4 in a 12-well plate. 4. On the day of transfection, trypsinize, harvest, and incubate SSCs cells on a gelatin-coated plate for 30–45 min at 37 °C, 5% CO2 to remove feeder cells. 5. Transfer SSCs into a 15-ml tube, and gently pipette SSCs into single cells. 6. Count cell number and collect a total of 2 × 10^6 cells for each transfection. Centrifuge SSCs at 100 × g for 5 min and discard supernatant. 7. Resuspend SSC pellet with 100 µl Nucleofector solution L, and add 10 µg total Cas9 plasmids to each cell suspension (see Note 7). 8. Transfer SSCs suspension into a transfection cuvette (sample must cover the bottom of the cuvette without air bubbles).
Place the cuvette into the Nucleofector holder and run program A020 by pressing the “X” button. 9. Immediately remove the cuvette from the Nucleofector, and add 500 µl of prewarmed SSC medium without penicillin and streptomycin into the cuvette. 10. Use the plastic pipette provided in the kit to carefully collect the cell suspension. Plate the suspension in a freshly prepared 6-well plate seeded with MEFs in SSC medium (penicillin and streptomycin-free).