Cas9 Nuclease, S. pyogenes

Product Information

    
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 nuclease, S. pyogenes, complexed with an sgRNA

In vitro DNA cleavage using Cas9 Nuclease, S. pyogenes

Product Source

A His-tagged engineered variant of E. coli that carries the cloned Cas9 gene from Streptococcus pyogenes.

This product is related to the following categories:

Genome Editing Products,

Programmable Nucleases Products

This product can be used in the following applications:

Genome Editing Applications

• Reagents Supplied

  The following reagents are supplied with this product:

  NEB https://www.neb.com/en/#	Component Name	Component https://www.neb.com/en/#	Stored at (°C)	Amount	Concentration
  M0386S     -20       Cas9 Nuclease, S. pyogenes M0386SVIAL -20 1 x 90 pmol 1 µM   NEBuffer™ r3.1 B6003SVIAL -20 1 x 1.25 ml 10 X
  M0386T     -20       Cas9 Nuclease, S. pyogenes M0386TVIAL -20 1 x 500 pmol 20 µM   NEBuffer™ r3.1 B6003SVIAL -20 1 x 1.25 ml 10 X
  M0386M     -20       Cas9 Nuclease, S. pyogenes M0386MVIAL -20 1 x 2,500 pmol 20 µM   NEBuffer™ r3.1 B6003SVIAL -20 2 x 1.25 ml 10 X

• Properties & Usage

  Reaction Conditions

  1X NEBuffer™ r3.1
  Incubate at 37°C

  1X NEBuffer™ r3.1
  100 mM NaCl
  50 mM Tris-HCl
  10 mM MgCl₂
  100 µg/ml Recombinant Albumin
  (pH 7.9 @ 25°C)

  Storage Buffer

  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 minutes

• Related Products

  Companion Products

  • EnGen® sgRNA Synthesis Kit, S. pyogenes
  • EnGen® Mutation Detection Kit

• Product Notes

  1. 1,000 nM is equal to 159 ng/μl.
  2. If planning to use higher concentration Cas9 Nuclease, S. pyogenes (M0386T and M0386M) for in vitro digestion of DNA, the enzyme should be diluted to 1 µM using Diluent B (NEB https://www.neb.com/en/#B8002S) prior to the reaction assembly.

• References

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

Protocols, Manuals & Usage

• 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 (https://www.neb.com/en/#M0386)

• Application Notes

  • Bridging dsDNA with a ssDNA Oligo and NEBuilder® HiFi DNA Assembly to create an sgRNA-Cas9 Expression Vector
  • Protocol for using Recombinant Cas9 Nuclease to Assess Locus Modification in Genome Editing Experiments

FAQs & Troubleshooting

• 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?
  6. How do I dilute the enzyme to 1 μM for in vitro reactions?
  7. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

Citations & Technical Literature

• Citations

  Product Citation Tool

  Additional Citations

  • Haussmann IU, Ustaoglu P, Brauer U, Hemani Y, Dix TC, Soller M. (2018) Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4 Nucleic Acids Res; Dec 12, PubMedID: 30541104, DOI: https://www.neb.com/en/10.1093/nar/gky1254
  • 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

• Feature Articles

  • CRISPR/Cas9 & Targeted Genome Editing: New Era in Molecular Biology

Quality, Safety & Legal

• Quality Control Assays

  Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

• Specifications & Change Notifications

  The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

  • M0386S_L_v1
  • M0386T_M_v1

• Certificate of Analysis

  The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

  • M0386M_v1_10011915
  • M0386T_v1_10011917
  • M0386S_v1_10018044
  • M0386M_v1_10028145
  • M0386T_v1_10028147
  • M0386S_v1_10035061
  • M0386M_v1_10043718
  • M0386T_v1_10043721
  • M0386M_v1_10057223
  • M0386T_v1_10057222
  • M0386S_v1_10057218
  • M0386M_v1_10062631
  • M0386T_v1_10062630
  • M0386M_v1_10084269
  • M0386M_v1_10091235
  • M0386T_v1_10084273
  • M0386S_v1_10084274
  • M0386M_v1_10102461
  • M0386T_v1_10114005
  • M0386S_v1_10121389
  • M0386M_v1_10128353
  • M0386S_v1_10129857
  • M0386T_v1_10125001
  • M0386M_v1_10144358
  • M0386T_v1_10146848
  • M0386S_v1_10149086
  • M0386S_v1_10171874
  • M0386T_v1_10171881
  • M0386M_v1_10174215
  • M0386T_v1_10209602
  • M0386S_v1_10211544
  • M0386M_v1_10212157
  • M0386S_L_v1_0081711
  • M0386T_M_v1_0081711
  • M0386M_v1_10238066
  • M0386T_v1_10243952
  • M0386S_v1_10253796
  • M0386M_v1_10263055
  • M0386T_v1_10280583

• Safety Data Sheets

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

  • Cas9 Nuclease, S. pyogenes
  • NEBuffer™ r3.1

• Legal and Disclaimers

  Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
  
  This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
  
  New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.