EnGen® Cas9 NLS, S. pyogenes

Description

EnGen Cas9 NLS, 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. EnGen Cas9 NLS, S. pyogenes 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:

Store at (°C)Concentration
Cas9 Nuclease Reaction Buffer-2010X

Properties and Usage

Reaction Conditions

1X Cas9 Nuclease Reaction Buffer
Incubate at 37°C

1X Cas9 Nuclease Reaction Buffer:
20 mM HEPES
100 mM NaCl
5 mM MgCl2
0.1 mM EDTA
pH 6.5 @ 25°C

Storage Temperature

-20°C

Storage Conditions

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 min

Notes

  1. 20 μM is equal to 3.22 mg/ml

References

  1. Jinek M. et al. (2012). Science. 816-821. DOI: 10.1126/Science.1225829. Epub 2012 Jun 28, PubMedID: 22745249

FAQs

  1. Where is the nuclear localization signal on EnGen® Cas9 NLS, S. pyogenes located?
  2. Which nuclear localization signal is fused to EnGen® Cas9 NLS, S.pyogenes?
  3. What is the difference between EnGen® Cas9 NLS, S. pyogenes (NEB #M0646) and Cas9 NLS, S.pyogenes (NEB #M0641)?
  4. Why do I observe incomplete digestion?
  5. Why does digestion efficiency differ between two different guide RNAs?
  6. Does NEB provide plasmids for gRNA cloning?

Protocols

  1. Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
  2. Electroporation of Cas9 RNP (ribonucleoprotein) into adherent cells using the Neon® Electroporation

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Feature Articles

Interactive Tools

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Functional Test (Cas9 Nuclease, Targeted Digestion):
    Cas9 Nuclease is tested in a reaction containing target DNA and guide RNA. The percent digested target DNA is determined by agarose gel electrophoresis.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
  • RNase Assay (Extended Digestion):
    The product is tested in a reaction containing a fluorescent RNA substrate. After incubation >90% of the substrate RNA remains intact as determined by gel electrophoresis.

Certificate of Analysis

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

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]

Safety Data Sheet

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

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.