2.7. Construction of the Cas9-Blast High-Expressed mESC Lineage

The lentivirus was packaged with lentiCas9-Blast plasmid, and used for wild-type mESC infection. The culture medium was changed after 8 hours of infection, and cells were further cultured for 48 hours. Then, Cas9-expressing cells were selected with 2 μg/ml blasticidin for 3 days. In order to get the high expression of Cas9 cell lines, those Cas9-expressing mESCs were seeded at an extremely low density, ensuring that each clone originated from a single cell. 3 days later, about 20 clones were chosen for expansion and establishment into the Cas9-expressing cell lines. The cell lines with the highest expression level of Cas9 mRNA were used for further infection with GeCKO-sgRNA library virus.