Product Class: Other

EnGen® Spy Cas9 NLS
neb31 cloned at NEB recombinant 37 65 Heat

Now available: EnGen Spy Cas9 HF1 NEB #M0667 for reduction of off-target cleavage

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.

Product Introduction

  • 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)
Catalog # Size Concentration
M0646T 500.0 pmol 20 µM
M0646M 2500.0 pmol 20 µM

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Product Information

Description

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.
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

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

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

Product Notes

  1. 20 μM is equal to 3.22 mg/ml

References

  1. Jinek M. et al. (2012). Science. 816-821. PubMedID: 22745249,

Protocols, Manuals & Usage

Protocols

  1. In vitro digestion of DNA with EnGen Spy Cas9 NLS (M0646)
  2. Protocol for Electroporation of EnGen® SpyCas9 NLS, S. pyogenes RNP (ribonucleoprotein) into adherent cells using the Lonza 4D-Nucleofector™ System
  3. Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
  4. Electroporation of EnGen® Spy Cas9 NLS RNP (ribonucleoprotein) into adherent cells using the Neon® Electroporation System
  5. In vitro digestion of plasmid DNA with EnGen SpRY Cas9 (NEB #M0669)

FAQs & Troubleshooting

FAQs

  1. Where is the nuclear localization signal on EnGen® Spy Cas9 NLS located?
  2. Which nuclear localization signal is fused to EnGen® Spy Cas9 NLS?
  3. What is the difference between EnGen® Spy Cas9 NLS (NEB #M0646) and Cas9 NLS, S.pyogenes (NEB #M0641)?
  4. Why do I observe incomplete digestion?
  5. Why does digestion/editing efficiency differ between two different guide RNAs?
  6. Does NEB provide plasmids for gRNA cloning?
  7. How do I dilute the enzyme to 1 μM for in vitro reactions?
  8. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

Quality, Safety & Legal

Quality Assurance Statement

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 DataSheets

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

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.

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