Product Class: Other

EnGen® Spy Cas9 Nickase
neb31 recombinant 37 65 Heat

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

  • Variant of Cas9 nuclease differing by a point mutation (D10A) in the RuvC nuclease domain
  • Capable of generating nicks, but not cleaving DNA
  • DNA double strand breaks can be generated, with reduced off-target cleavage, by targeting two sites with EnGen Cas9 Nickase in close proximity
Catalog # Size Concentration
M0650S 90.0 pmol 1 µM
M0650T 500.0 pmol 20 µM

Product Information

Description

EnGen® Spy Cas9 Nickase is a variant of Cas9 nuclease differing by a point mutation (D10A) in the RuvC nuclease domain, which enables it to nick, but not cleave, DNA (1,2). EnGen Spy Cas9 Nickase, like EnGen Cas9 NLS (NEB #M0646), targets DNA using a guide RNA complementary to a site with a 3’ NGG protospacer adjacent motif (PAM). Nicking occurs on the DNA strand opposite the PAM. Double-stranded DNA breaks can be generated with reduced off-target cleavage by targeting two sites in close proximity (generally 0-20 bp apart) and with PAMs facing outward to leave 5’ overhangs. Nicking by EnGen Spy Cas9 Nickase occurs on the DNA strand opposite the target sequence. EnGen Spy Cas9 Nickase contains SV40 T antigen nuclear localization (NLS) sequences on the N and C- termini.

Product Source

EnGen® Spy Cas9 Nickase is expressed as a N-terminal 6XHis-tagged fusion in E. coli.
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

Application Features

  • Paired, offset cleavage of genomic loci
  • Target-specific DNA nicking

Product Notes

  1. 20 µM is equal to ~3.2 mg/ml EnGen® Cas9 Nickase NLS, S. pyogenes. 1 µM is equal to ~0.16 mg/ml.
  2. This product is compatible with Diluent Buffer B: 300 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 500 µg/ml BSA and 50% glycerol. (pH 7.4 @ 25°C).

References

  1. Mali, P., et. al. (2013). NatBiotech. 31 (9), 838-8.
  2. Ran, FA., et. al. (2013). Cell. 154 (6), 1380-9.

Protocols, Manuals & Usage

Protocols

  1. In vitro digestion of DNA with EnGen Spy Cas9 Nickase (M0650)

FAQs & Troubleshooting

FAQs

  1. What is the difference between EnGen® Spy Cas9 Nickase (NEB #M0650) and EnGen Spy Cas9 NLS (NEB #M0646)?
  2. Which nuclear localization signal is fused to EnGen® Spy Cas9 Nickase?
  3. Why does binding efficiency differ between two different guide RNAs?
  4. Does NEB offer synthetic sgRNAs or plasmids for cloning?
  5. How should offset sgRNAs be oriented for efficient modification using EnGen® Spy Cas9 Nickase?
  6. Why do I observe incomplete digestion?
  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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