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CRISPRcraft™ S.p. Cas9 Nuclease

Cat.No. Size Price Quantity Subtotal Buy
70020-1 120 ug 來電價
70020-2 400 ug 來電價

Products Description

特色:

  • 重組wild-type Streptococcus pyogenes Cas9 nuclease, 蛋白C端含進核訊號(NLS)序列,確保有效遞送至細胞核
  • 高品質蛋白 (QC品質保證:  Purity > 95%, Endotoxin < 10  EU/mg)
  • 濃度高 (10mg/mL, 62 µM)配合多種轉染條件
  • 另有Control kit,可用於In vivo和In vitro實驗中酵素活性測試

CRISPR-Cas9 gene editing is based on activity of Cas9 nuclease, an RNA-guided endonuclease (RGEN). Cas9 catalyzes sequence-specific cleavage of double-stranded DNA by forming a ribonucleoprotein (RNP) complex with a small (~97 nt) guide RNA that directs Cas9 to the desired locus. This precise DNA cleavage enables a variety of in vivo and in vitro applications including gene editing, CRISPR-mediated DNA detection, sequence enrichment and depletion.
CRISPRcraft™ S.p. Cas9 Nuclease is a purified recombinant wild-type Streptococcus
pyogenes Cas9 nuclease produced in E. coli, containing a C-terminal 6xHis tag and a C-terminal NLS to allow efficient transport to the nucleus. It is provided at high concentration (10 mg/mL, 62 µM) to enable efficient RNP delivery and compatibility with multiple delivery methods including lipid-based transfection and electroporation (Figures 1 and 2).

 
Figure 1: CRISPR RNP delivery.
HEK293T cells were transfected in triplicate with RNP containing CRISPRcraft™ S.p. Cas9 Nuclease and guide components from the CRISPRcraft™ S.p. Cas9 Nuclease Control Kit targeting HPRT, using either lipofection or electroporation. Transfection conditions: TransIT-X2® Dynamic Delivery System (Mirus), 50 nM gRNA and 25 nM Cas9; Lipofectamine® RNAiMAX (ThermoFisher Scientific), equimolar Cas9 and gRNA at 25 nM; Ingenio® Electroporation Solution (Mirus), 1.5 µM gRNA and 0.75 µM Cas9, plus Alt-R® Cas9 Electroporation Enhancer (IDT). At 48 hours post-transfection, cleavage efficiency at the HPRT locus was determined using the T7E1 mismatch detection assay and is expressed as % gene modification.
 
Figure 2: In vivo gene editing with different guide RNA sequences.
HEK293T cells were independently transfected in triplicate with RNP containing CRISPRcraft™ S.p. Cas9 Nuclease and three different guides, each targeting a different loci within human HPRT. Transfections were performed using TransIT-X2® Dynamic Delivery System (Mirus), 20 nM guide and 10 nM Cas9 (2:1 guide:Cas9 molar ratio). Cleavage efficiency at each HPRT locus was determined using the T7E1 mismatch detection assay, 48 hours post-transfection.


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Information

Assay Acceptance Criteria
Purity ≥ 95%, by SDS-PAGE
Endotoxin ≤ 10 EU/mg, by LAL
Endonuclease Activity < 10% nicking as determined by agarose gel electrophoresis, 150 ng S.p. Cas9 per assay.
Exonuclease Activity No detectable DNA degradation as determined by agarose gel electrophoresis, 150 ng S.p. Cas9 per assay.
RNase Activity < LOD, by fluorescence-based assay, 15 µg S.p. Cas9 per assay (detection limit equivalent to 0.5 pg RNase A).
DNA Contamination < LOD, by agarose gel electrophoresis.
Activity Assay < 80% of linearized target DNA at 2 nM is digested with 20 nM S.p. Cas9 with equal molar ratio of guide RNA at 37°C for 10 min.

Resources

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