The Cas9-NLS protein is a recombinant endonuclease from Streptococcus pyogenes fused from the C-terminus to the repeated nuclear localization signal (NLS) of the SV40 virus (PKKKRKV). The molecular weight of the Cas9-NLS protein is163 kDa. Cas9-NLS endonuclease in complex with RNA guides (crRNA duplex: tracrRNA) or single sgRNA catalyzes site-specific hydrolysis of the phosphodiester bond in double-stranded DNA (Figure). The cleavage is on the DNA chain between the third and fourth nucleotides from the PAM sequence (NGG is a motif adjacent to the protospacer) with the formation of blunt ends.
Figure: DNA digestion with sgRNA:Cas9-NLS complex.
Source: Cas9-NLS endonuclease purified from E. coli strain containing a plasmid with the cloned full length gene of Cas9 Streptococcus pyogenes.
Quality control. Each batch of the enzyme is tested for enzyme activity, electrophoretic purity, and the absence of non-specific endonuclease activity. The purity of the enzyme is ≥95% (SDS-PAGE), nonspecific endonuclease activity is absent.
Storage buffer: 300 мМ NaCl, 50 мМ Tris-HCl, 0,1 мМ EDTA, 5 мМ β-mercaptoethanol, 50% glycerol (pH=7.5 @ 25°C).
Standard buffer for the plasmid DNA hydrolysis reaction: 20 mM HEPES, 125 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 6 mM MgCl2, 7% glycerol (pH 7.5 @ 25 ° C).
The optimum reaction temperature: 37°C.
Enzyme inactivation: 65°C for 5 minutes.
Standard product concentration: 20 μM (20 pmol/μl)
Packing unit: 300 pmoles (15 μl) and 500 pmoles (25 μl)