EnGen Cas9 NLS, S. pyogenes, is an RNA-guided endonuclease that catalyzes site- specific cleavage of double stranded DNA. The location of the break is within the target sequence 3 bases from the NGG PAM (Protospacer Adjacent Motif) (1). The PAM sequence, NGG, must follow the targeted region on the opposite strand of the DNA with respect to the region complementary sgRNA sequence. EnGen Cas9 NLS, S. pyogenes contains Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the N- and C- termini of the protein.