Cas9 Nuclease, S. pyogenes

Description

sgRNA Designer Tool














    
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 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.)


Cas9 nuclease, S. pyogenes, complexed with an sgRNA Schematic representation of Cas9 Nuclease, S. pyogenes sequence recognition and DNA cleavage
Cleavage occurs three nucleotides upstream of the PAM sequence (shown in red). sgRNAs are complimentary to the strand of DNA opposite of the PAM.


In vitro DNA cleavage using Cas9 Nuclease, S. pyogenes 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

Heat Inactivation

65°C for 5 min

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?
  4. Does Cas9 Nuclease contain a nuclear localization signal (NLS)? 
  5. Is Cas9 Nuclease fused with a N-terminal 6XHis tag?

Protocols

  1. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
  2. Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (NEB #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

Interactive Tools

NEB Publications

  • Pardee P, Green A A, Takahashi M K, Braff D, Lambert G, Lee J W, Ferrante T, Ma D, Donghia N, Fan M, Daringer N M, Bosch I, Dudley D M, O’Connor D H, Gehrke H, Collins J J (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components Cell. DOI: 10.1016/j.cell.2016.04.059

Citations

  • Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, Lockey RF (2015). CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning Biotechniques. 58(4), 161-70. PubMedID: 25861928, DOI: 10.2144/000114261
  • Lee, NC., Larionov, V., Kouprina, N. (2015). Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast Nucleic Acids Res. 43(8), e55. PubMedID: 25690893

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. Further information regarding NEB product quality can be found here.
  • 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.

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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.