Cas9 Nuclease Protein

CAT.NOUNITPRICE
K0088.0µg (50pmol/ 50µL)
$25.00

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Cas9 Nuclease Protein
Cas9 Nuclease Protein

In stock

$25.00

Summary
    Specifications


    Description

    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.

    The Cas9 nuclease serves to unwind the genomic DNA duplex next to conserved protospacer adjacent motifs (PAMs) and homes in on its target sequence, which is recognized by a complementary single-guide RNA. The resulting double-stranded break gets repaired by the non-homologous end joining (NHEJ) pathway, leading to a disruption in the open reading frame of the targeted gene. Alternatively, by supplying a suitable repair template, virtually any desired point mutation can be introduced at the break point via homology-directed repair (HDR).

    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.

    SKUK008
    Cas TypeNuclease
    Cas OriginspCas9
    Cas Protein MarkerNo GFP
    Source

    E. coli

    AliasspCas9, spyCas9, CRISPR-associated endonuclease Cas9 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.
    Documents


    Supporting Protocol
    MSDS

      QC

        Other
        FAQs


        Does K008 contain a HIS-tag?
        Yes, it does contain a His-tag.
        Does this cas9 nuclease protein contain a Nuclear Localization Signal?
        No, this Cas9 nuclease protein does not have a Nuclear Localization Signal and therefore represents the native form. If NLS is desired, we would recommend our Cas9 Nuclease NLS Protein (Cat. No. K030, K130). A full list of our Cas9 products can be found here:
        https://www.abmgood.com/CRISPR-Cas9-Nuclease-Nickase-Vectors-Viruses.html
        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.
        References


        1
        • 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.