1. Seed mESCs on 6-well plate with LIF-supplemented medium (Day 1). 2. Co-transfect 1 μg SPH, 1 μg OminiCMV-mCherry, and 0.6 μg PBase using Lipofectamine 3000. Replace medium after 12 hours (Day 2). 3. Digest mESCs with 0.05% trypsin for 4 minutes at 37 °C, inactivate trypsin, centrifuge at 1000 rpm for 3 min, seed to new 6-well plates, treat with puromycin (1 μg/ml) for 4-5 days (Day 4). 4. Digest mESCs with 0.05% trypsin, inactivate trypsin, centrifuge at 1000 rpm for 3 min, seed to new 6-well plate (Day 8). 5. Digest mESCs with 0.05% trypsin, prepare for FACS into 96-well plates (Day 10). 6. Remove single colonies from 96-well plates to 24-well plates, confirm positive colonies by transient transfection of sgRNAs and PCR analysis (SPH and OminCMV primers) (Day 14-15). 7. Seed SPH-OminiCMV mESCs on 12-well plates with LIF-supplemented medium (Day 1). 8. Co-transfect 0.5 μg SaKKHCas9-GFP-sgRNA and 1 μg SpCas9 sgRNA precursor using Lipofectamine 3000. Replace medium after 12 hours (Day 2). 9. Digest mESCs with 0.05% trypsin, prepare for FACS, sort positive cells, seed GFP positive cells into 12-well plates (Day 4). 10. Digest mESCs with 0.05% trypsin, seed to new 12-well plates (Day 8). 11. Sort single cells into 96-well plates by FACS, confirm insertion by PCR (Day 18). 12. Remove single colonies from 96-well plates to 24-well plates, confirm positive colonies by PCR (Day 22). 13. Measure fluorescent intensity of colonies by FACS, take fluorescence images under confocal microscope (Day 27).