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EnGen® Spy Cas9 NLS

Catalog # Concentration Size List Price Quantity Your Price
M0646M 20 µM 2500 pmol $914.00
$822.60
M0646T 20 µM 500 pmol $229.00
$206.10
Catalog # Size List Price Your Price
M0646M 2500 pmol $914.00
$822.60
M0646T 500 pmol $229.00
$206.10
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
  • Ideal for direct introduction of Cas9/sgRNA complexes
  • Dual NLS for improved transport to the nucleus
  • Compatible with EnGen® sgRNA Synthesis Kit, S. pyogenes (NEB #E3322S) and the EnGen Mutation Detection Kit (NEB #E3321S)
Featured Videos
View Video Library
  • Monarch_MiniprepProtocol_VideoThumb
    Bacterial Adaptive Immunity with CRISPR/Cas9
  • Monarch_MiniprepProtocol_VideoThumb
    NEB® TV Ep. 7 – Synthetic Biology/ CRISPR
EnGen Spy Cas9 NLS, 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. EnGen Spy Cas9 NLS contains Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the N- and C- termini of the protein.

Increased genome editing efficiency using Cas9 RNP delivery

Direct
Cas9 and sgRNA targeting a human gene were delivered to HEK293 cells by transfection. Transfected plasmid DNA contained expression cassettes for 2x NLS (N- and C-terminal) Cas9 and sgRNA. Plasmid DNA was delivered using TransIT-X2 (Mirus). Transfected mRNA was modified with pseudouridine and 5-methylcytosine and encoded 2x NLS (N-and C-terminal) Cas9. sgRNA was co-transfected with the mRNA using TransIT-mRNA. Cas9 RNPs were delivered in reverse transfections using Lipofectamine RNAiMAX (Life Technologies) using 10 nanomolar final concentration of ribonucleoprotein (RNP). Cas9 has no NLS in the protein sequence. EnGen Cas9 has N- and C-terminal NLSs. The efficiency of editing was determined using T7 Endonuclease I assay and is expressed as % modification.
Product Source
An E. coli strain that carries the cloned Cas9 gene from Streptococcus pyogenes with N- and C-terminal Simian virus 40 (SV40) nuclear localization signals (NLS) and a N- terminal 6XHis tag.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  EnGen® Spy Cas9 NLS M0646TVIAL -20 1 x 500 pmol 20 µM
  NEBuffer™ r3.1 B6003SVIAL -20 1 x 1.25 ml 10 X
  EnGen® Spy Cas9 NLS M0646MVIAL -20 1 x 2,500 pmol 20 µM
  NEBuffer™ r3.1 B6003SVIAL -20 1 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

Notes
  • 20 μM is equal to 3.22 mg/ml
References
Quality Control Assay
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
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]
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]
Safety Data Sheets
Legal And Disclaimer

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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.

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