Recombinant S. pyogenes CRISPR-associated Protein 9, CF

R&D Systems, part of Bio-Techne | Catalog # 9957-C9

Purified CAS9 with Nuclear Localization Sequence
R&D Systems, part of Bio-Techne
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9957-C9-050
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Key Product Details

Source

E. coli

Conjugate

Unconjugated

Applications

Enzyme Activity

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Product Specifications

Source

E. coli-derived s. pyogenes CRISPR-Cas9 protein
APKKKRKVGIHGVPAA S. pyogenes CRISPR-Cas9
(Asp2-Asp1368)
Accession # Q99ZW2		 KRPAATKKAGQAKK-KKGYGRKKRRQRRRG HHHHHH
N-terminus C-terminus

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Ala

Predicted Molecular Mass

164 kDa

SDS-PAGE

133 kDa, reducing conditions

Activity

Measured by its ability to cleave a targeted DNA substrate.
S. pyogenes CRISPR-Cas9 achieves >80% substrate cleavage, as measured under the described conditions.

Scientific Data Images for Recombinant S. pyogenes CRISPR-associated Protein 9, CF

Recombinant S. pyogenes CRISPR-associated Protein 9 Bioactivity

Recombinant S. pyogenes CRISPR-associated Protein 9 Bioactivity

Quantitative analysis of the substrate cleavage by densitometry of the agarose gel. Error bars display standard error of 3 replicates. 9957-C9 has equivalent activity to a Leading Competitor's Cas9 using the insert assay protocol.
Recombinant S. pyogenes CRISPR-associated Protein 9 Bioactivity

Recombinant S. pyogenes CRISPR-associated Protein 9 Bioactivity

Agarose gel image of in vitro cleavage of DNA substrate into two DNA products by 9957-C9 and a Leading Competitor's Cas9.
Recombinant S. pyogenes CRISPR-associated Protein 9 SDS-PAGE

Recombinant S. pyogenes CRISPR-associated Protein 9 SDS-PAGE

2 μg/lane of Recombinant S. pyogenes CRISPR-Cas9 was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at ~130 kDa.

Formulation, Preparation and Storage

9957-C9
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, Glycerol and TCEP.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: CRISPR-Cas9

Streptococcus pyogenes Cas9 (CRISPR associated protein 9) is a 160 kDa RNA guided endonuclease that introduces site specific cleavage of double strand DNA (1). It is part of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system found in many bacteria such as S. pyogenes and most archaea, which provide adaptive immunity against invading mobile genetic elements (such as viruses, transposable elements and conjugative plasmids) (2, 3). Upon viral infection, short viral DNA (known as "spacers") integrate into the host genome between CRISPR repeats, and RNA sequences (guide RNA or gRNA) with this genetic information help guide Cas9 protein to recognize and cut foreign DNA. Cas9 protein undergoes conformational changes upon gRNA binding that shift from non-DNA binding conformation into an active DNA binding conformation. In the Cas9-gRNA complex, the gRNA sequence remains accessible to interact with free DNA, and the extent to which the gRNA spacer and target DNA segment (known as "protospacer") match will determine the cut site (4). The presence of a 5′-NGG-3′ protospacer adjacent motif (PAM) sequence immediately downstream of protospacers is required for Cas9 cleavage of the foreign DNA. PAM is absent in bacterial CRISPR loci, therefore preventing cleavage of the host genome (4). Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM specificity to this process (5). This RNA guided nuclease system called CRISPR/Cas (CRISPR associated protein) has been widely applied to genome engineering with increased efficiency (6). The attached nuclear localization signals(NLSs) on the chimeric protein ensures nuclear compartmentalization in mammalian cells during gene editing (7).

References

  1. Feng, Z. et al. (2013) Cell 154:1380.
  2. Moineau. et al. (2010) Nature 468:67.
  3. Barrangou, R. et al. (2014) Molecular Cell 54:234.
  4. Charpentier, E. et al. (2011) Nature 471:602.
  5. Heler, R. et al. (2015) Nature 519:199.
  6. Thomson, J.A. et al. (2013) PNAS,110:15644.
  7. Cong, L. et al. (2013) Science 339:819.

Long Name

CRISPR-associated Protein 9

Alternate Names

Cas9, CRISPR-associated endonuclease Cas9/Csn1, CRISPR-Cas9/Csn1, CRISPR/Cas9, csn1, SPy_1046, SPy1046, SpyCas9

Entrez Gene IDs

901176 (S. pyogenes)

Gene Symbol

CAS9

Additional CRISPR-Cas9 Products

Product Documents for Recombinant S. pyogenes CRISPR-associated Protein 9, CF

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Product Specific Notices for Recombinant S. pyogenes CRISPR-associated Protein 9, CF

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