| Description |
Ultra eSpCas9-2NLS Nuclease (10 mg/ml) is a mutant form of Cas9 nuclease and is produced by expression in an E. coli strain carrying a plasmid encoding the eSpCas9 gene from Streptococcus pyogenes with double-ended nuclear localization signal (NLS). Enhanced specificity SpCas9 (eSpCas9), also referred to as SpCas9 (K848A/K1003A/R1060A), is structurally engineered for improved target specificity by researchers at the Feng Zhang Laboratory at the Broad Institute (Slaymaker et al. 2016). Ultra eSpCas9-2NLS is able to reduce off-target effects by over 10-fold, while maintaining robust on-target genome editing efficiency. Cas9 genome editing is dependent on the separation of DNA double strands. Mismatches between sgRNA and untargeted DNA sequences can cause unspecific binding and cleavage. To improve genome editing specificity, SpCas9 with mutations K848A, K1003A, and R1060A was developed. Neutralization of these positively charged residues within the non-target strand groove of SpCas9 weakened non-target binding and encouraged on-target binding which requires more stringent Watson-Crick base pairing. Features: The protein is free from additional tagging amino acid. Supplied as a solution of 25 mM Tris, 300 mM NaCl, 0.1 mM EDTA, 50% Glycerol pH 8.0 at 25°C. MW: ~160kDa Active Temperature: 37°C. Cas9 protein forms a very stable ribonucleoprotein (RNP) complex with the guide RNA (gRNA) component of the CRISPR/Cas9 system. The Cas9 RNP complex can localize to the nucleus immediately upon entering the cell with the addition of a nuclear localization signal (NLS), and catalyze vleaveage of double stranded DNA. There is no requirement for transcription and translation compared with mRNA or plasmid systems. Additionally, the Cas9 RNP complex is rapidly cleared from the cell minimizing the chance of off-target cleavage when compared to other systems (Kim, et al. 2014). This DNA-free system avoids the risk of inserting foreign DNA into the genome, which can be quite useful for gene editing-based disease therapy. GenScript has developed a NLS-Cas9-NLS nuclease which contains a nuclear localization sequence (NLS) on both ends of the protein to meet all the researchers’ requirements. To order full size Cas9 protein, please order through https://www.genscript.com/cas9-ultra-enzymes.html |
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| Concentration | 10 mg/ml | ||||||||||||||||
| Storage Condition | Stable for at least 12 months at -20°C. Avoid repeated freeze-thaw cycles. | ||||||||||||||||
| Quality Specifications |
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