Cas9 is a protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses. Cas9 has been widely used for gene editing in mammalian cells. eSpCas9 (enhanced specificity Cas9) has enhanced specificity compared to wild type Cas9 for CRISPR/Cas9 genome editing. eSpCas9 is able to reduce off-target effects and maintain robust on-target genome editing efficiency, allowing more efficient gene targeting. Tet-inducible Cas9 lentivirus (CAG-rtTA, Puro) expresses eSpCas9 under the control of the tetracycline-responsive element (TRE) with CAG promoter diven puromycin as a selection marker, allowing selecting stable cell clones with puromycin.