Cas9 Nickase D10A NLS Protein

K0348.0µg (50pmol/ 50µL)

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Cas9 Nickase D10A NLS Protein
Cas9 Nickase D10A NLS Protein

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    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is the latest RNA-guided, endonuclease tool in genome editing which allows for very specific genomic disruption and replacement. One concern with the current CRISPR Cas9 technology is the potential off-target effects of the Cas9 nuclease.

    To counteract off-target mutagenic effects of this system, the Cas9 Nickase D10A was developed with a D10A mutation in its RuvC1 nuclease domain. Unlike the Cas9 nuclease, this mutant form generates a single-stranded nick instead of a double-strand break (DSB). Because a single DNA nick is quickly repaired with high fidelity by the cellular machinery, the system requires two closely juxtaposed nicks in order to trigger the same genomic disruption as the Cas9 nuclease. This effectively boosts the recognition sequence to 40 instead of 20 nucleotides, and, as a result, off-target effects become highly unlikely. Thus, the double-nickase CRISPR system offers unparalleled specificity to satisfy even the most stringent of experimental requirements.

    The Cas9 nuclease from the bacteria Streptococcus pyogenes, abbreviated spCas9, is the most commonly used Cas9 variant. The reason for spCas9 popularity is two-fold. First, the spCas9 PAM sequence is 5’-NGG, which is highly abundant in the genome allowing virtually any gene to be targeted. The spCas9 enzyme also has on average a higher efficiency in vivo compared to other variants. Cas9 D10A Nickase NLS contains a SV40 T antigen nuclear localization sequence (NLS) on the C-terminus of the protein.

    Cas TypeNickase
    Cas OriginspCas9
    Cas Protein MarkerNo GFP

    E. coli

    AliasCas9n, spCas9n, Cas9D10A, Cas9(D10A), CRISPR-associated endonuclease Cas9(D10A) from Streptococcus pyogenes
    Unit quantity8.0µg (50pmol/ 50µL)
    Product Concentration1000 nM, 160 µg/ml
    FormatEnzyme supplied with 10X Reaction Buffer
    Storage Buffer10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 300 mM NaCl, and 50% (v/v) Glycerol.
    StorageStore all components at -20°C.
    CautionThis product is distributed for laboratory research only. Caution: Not for diagnostic use.

    Does K034 contain a HIS-tag?
    Yes, it does contain a His-tag.
    What is the in vitro digestion of DNA protocol with Cas9 Nucleases?
    1. 1. Mix individual components before use and assemble reaction at room temperature.
      Component Volume
      sgRNA (300 nM) 3 µl
      Cas9 Nuclease Protein (1 µM) 1 µl
      10X Cas9 Reaction Buffer 3 µl
      Nuclease-free H2O 20 µl
      Pre-incubate without substrate for 15 min at 37°C
      Substrate DNA (30 nM) 3 µl
      Total Volume 30 µl

    2. 2. Mix thoroughly, and pulse-spin in a microfuge.
    3. 3. Incubate at 37°C for 1 hour.
    4. 4. (Optional) Add 1 μl of Proteinase K to each sample. Mix thoroughly and pulse-spin in a microfuge. Incubate at room temperature for 10 minutes.
    5. 5. Analyze fragments via agarose gel electrophoresis.

    The substrate DNA: sgRNA : Cas9 molar ratio must be kept at 1:10:10 for highest efficiency.

    • Nelson, C. E., Wu, Y., Gemberling, M. P., Oliver, M. L., Waller, M. A., Bohning, J. D., … Gersbach, C. A. "Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy" Nature Medicine 25(3):427–432 (2019). DOI: 10.1038/s41591-019-0344-3.