Product Class: Other

EnGen® Seq1 Cas9
neb31 recombinant 37 65 Heat

Product Introduction

  • 5´- NAGA -3´ PAM sequence allows targeting of additional genomic regions
  • Ideal for direct introduction of Cas9/sgRNA complexes
  • Compatible with the EnGen Mutation Detection Kit (NEB #E3321S)
  • Active in in vitro reactions from 20°C to 45°C
Catalog # Size Concentration
M0668T 500.0 pmol 20 µM

Product Information

Description

EnGen Seq1 Cas9 from Streptococcus equinus is an RNA-guided DNA endonuclease that catalyzes site-specific cleavage of double-stranded DNA (dsDNA). Targeting requires the formation of a ribonucleoprotein complex of Cas9 and a 116-nucleotide single guide RNA (sgRNA). The sgRNA encodes a 20-nucleotide sequence with complementarity to the DNA target strand immediately upstream of a 5´– NAGA –3´ protospacer adjacent motif (PAM) on the non-target strand. DNA cleavage by EnGen Seq1 Cas9 produces a double-stranded break occurring 3 nucleotides upstream of the PAM. EnGen Seq1 Cas9 has Simian virus 40 (SV40) T antigen nuclear localization signals (NLS) at both the N- and C-termini of the protein.

Figure 1. Schematic representation of S. equinus Cas9 nuclease complexed with a single guide RNA and target DNA

Diagram of a sequence

S. equinus Cas9 (Seq1 Cas9) protein is shown in beige, single guide RNA (sgRNA) is depicted in blue, and the DNA is shown in grey, green, and red. The DNA target, or protospacer, is shown in green and the base pairing complementarity of the target strand with the guide RNA in the R-loop is illustrated. The protospacer adjacent motif (PAM) which is required for Cas9 binding to DNA is shown in red and the locations of DNA strand cleavage are indicated with black triangles.

Product Source

An E. coli strain that carries the cloned Cas9 gene from Streptococcus equinus with N- and C-terminal Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) and a C-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 3.26 mg/ml

Protocols, Manuals & Usage

Protocols

  1. In vitro digestion of DNA with EnGen® Seq1 Cas9 (NEB #M0668)

FAQs & Troubleshooting

FAQs

  1. Where is the nuclear localization signal on EnGen® Seq1 Cas9 located?
  2. Which nuclear localization signal is fused to EnGen® Seq1 Cas9?
  3. What is the difference between EnGen® Seq1 Cas9, EnGen Sau Cas9 (NEB #M0654), and EnGen Spy Cas9 NLS (NEB #M0646)?
  4. Why do I observe incomplete digestion/editing?
  5. Why does digestion/editing efficiency differ between two different single guide RNAs?
  6. Does NEB provide plasmids for sgRNA cloning?
  7. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?
  8. Can mutations generated with EnGen Seq1 Cas9 be detected using T7 Endonuclease I (NEB #M0302) or the EnGen Mutation Detection Kit (NEB #E3321S)?
  9.  Can sgRNA for use with EnGen Seq1 Cas9 be generated using the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322)?
  10. How do I design a single guide RNA for use with EnGen Seq1 Cas9?
  11. How do I dilute the enzyme to 1 μM for in vitro reactions?

Quality, Safety & Legal

Quality Assurance Statement

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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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