Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids (1). The CRISPR system consists of a short non-coding guide RNA (sgRNA) made up of a target complementary CRISPR RNA (crRNA) and an auxiliary transactivating crRNA (tracrRNA). The sgRNA guides the Cas9 endonuclease to a specific genomic locus via base pairing between the crRNA sequence and the target sequence, and cleaves the DNA to create a double-strand break. 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 (Fig.1).
Intact Genomics Cas9 Nuclease is the purified recombinant Streptococcus pyogenes Cas9 enzyme containing a nuclear localization signal (NLS) at the C-terminal for targeting to the nucleus. This enzyme is designed to perform CRISPR/Cas9-mediated genome editing (1, 2). The physical purity of this enzyme is ≥98% as assessed by SDS-PAGE with Coomassie® blue staining (Fig. 2).
- coli BL21 (DE3) strain expressing a Cas9 gene from Streptococcus pyogenes with an N-terminal 6xHis tag and C-terminal SV40 nuclear localization signal (NLS).
- Cas9 Nuclease
- 10x Cas9 Nuclease Reaction Buffer
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 °C
1x Cas9 Reaction Buffer
20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH 6.5 @ 25 °C
Quality Control Assays
Cas9 nuclease is free from detectable RNase, Endonuclease (nicking) and non-specific DNase activities.3272 3273 3276