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    Cas9 Nuclease, S. pyogenes

    Description


        
    Cas9 Nuclease, S. pyogenes, is an RNA-guided endonuclease that catalyzes site-specific cleavage of double stranded DNA. 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.

    Cas9 in vivo: Bacterial Adaptive Immunity
    CRISPR loci in the bacterial genome are transcribed and processed into crRNA. Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)


    Schematic representation of Cas9 Nuclease, S. pyogenes sequence recognition and DNA cleavage


    In vitro DNA cleavage using Cas9 Nuclease, S. pyogenes
    Reactions were set up according to recommended conditions, and cleavage products were resolved on a 1% agarose TBE gel. Input DNA is PvuII-linearized pBR322 plasmid DNA.

    Product Source

    An E. coli strain that carries the cloned Cas9 gene from Streptococcus pyogenes

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Cas9 Nuclease Reaction Buffer-2010X

    Properties and Usage

    Reaction Conditions

    1X Cas9 Nuclease Reaction Buffer
    Incubate at 37°C

    1X Cas9 Nuclease Reaction Buffer:
    20 mM HEPES
    100 mM NaCl
    5 mM MgCl2
    0.1 mM EDTA
    pH 6.5 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    300 mM NaCl
    10 mM Tris-HCl
    0.1 mM EDTA
    1 mM DTT
    50% Glycerol
    pH 7.4 @ 25°C

    Notes

    1. 1,000 nM is equal to 159 ng/μl.

    References

    1. Jinek M. et al. (2012). Science. 816-821 doi: 10.1126/Science.1225829. Epub 2012 Jun 28.. PubMedID: 22745249

    FAQs

    1. Why do I observe incomplete digestion?
    2. Why does digestion efficiency differ between two SgRNAs?
    3. Does NEB provide plasmids for gRNA cloning?

    Protocols

    1. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Feature Articles

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Functional Test (Cas9 Nuclease, Targeted Digestion):
      Cas9 Nuclease is tested in a reaction containing target DNA and guide RNA. The percent digested target DNA is determined by agarose gel electrophoresis.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Assay (Extended Digestion):
      The product is tested in a reaction containing a fluorescent RNA substrate. After incubation >90% of the substrate RNA remains intact as determined by gel electrophoresis.

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.