Cas9 nuclease, from Streptococcus pyogenes, is an RNA-guided endonuclease that catalyzes a site-specific cleavage of double stranded DNA. The cleavage occurs within the target sequence 3 bases from the NGG PAM (Protospacer Adjacent Motif). The PAM sequence must follow the targeted region on the opposite strand of the DNA with respect to the region complementary sgRNA sequence. Transfer of a custom-designed crRNA molecule for target gene together with tracrRNA and Cas9 will lead to a specific cleavage of target sequence in cells.dCas9, two points mutant (D10A and H840A) of wild-type Cas9 nuclease, is a catalytically inactive form of Cas9 and has a simple DNA binding activity. dCas9 contains a single nuclear localization sequence (NLS) at the C-terminus of the protein.dCas9 NLS is an optimized recombinant enzyme, which is expressed and purified from E.coli strain as a 6xHis-tagged form at the N-terminal end.
Applications- Genome editing Reagents Supplied & Storage Condition- dCas9 nuclease : 0.5 ㎍/㎕, Store at -20°C.- 10x Cas9 Reaction Buffer : Store at 4°C. Reaction Condition- 1X Cas9 Reaction Buffer. Incubate at 37°C. 10x Reaction Buffer- 500 mM Tris-HCl (pH 7.9), 1 M NaCl, 100 mM MgCl₂, 10 mM DTT. Storage Buffer- 10 mM Tris-HCl (pH 7.5), 50% glycerol, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT. QC Tests
- Activity, exo and endonuclease activity test, SDS-PAGE purity, performance tests.