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. eSpCas9 (CAG, Neo) lentivirus expresses eSpCas9 under the control of the CAG promoter with neomycin as a selection marker, allowing selecting stable cell clones with neomycin.