Recombinant S. pyogenes CRISPR-Cas9 protein (ab224892)

Description

  • Product name

    Recombinant S. pyogenes CRISPR-Cas9 protein
    See all CRISPR-Cas9 proteins and peptides
  • Purity

    > 95 % SDS-PAGE.
    Affinity purified using Ni-based resin.
  • Endotoxin level

    < 1.000 Eu/µg
  • Expression system

    Escherichia coli
  • Accession

  • Protein length

    Full length protein
  • Animal free

    No
  • Nature

    Recombinant
    • Species

      Streptococcus pyogenes
    • Additional sequence information

      Contains N-terminal and C-terminal Nuclear Localization Signal (NLS).

Specifications

Our Abpromise guarantee covers the use of ab224892 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

  • Applications

    SDS-PAGE

  • Form

    Liquid
  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

    Shipped at 4°C. Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.

    pH: 7.5
    Constituents: 0.02% DTT, 0.003% EDTA, 50% Glycerol, 1.74% Sodium chloride, 0.16% Tris HCl

General Info

  • Alternative names

    • Cas9
    • CRISPR-associated endonuclease Cas9/Csn1
    • CRISPR-Cas9/Csn1
    • csn1
    • SpyCas9
    see all
  • Relevance

    [FUNCTION] CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA) (Probable). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.

Images

  • In vitro DNA cleavage assay using ab224892. A Cas9 reporter plasmid linearized with PvuI was incubated with (lanes 2 and 3) or without (lane 1) ab224892 for 1 hour at 37°C. Lane 1 shows the untreated plasmid. A negative control reaction that lacked sgRNA is shown in lane 3. Cleavage efficiency was assessed by agarose gel electrophoresis.

  • In vitro transfection assay using ab224892. HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells were transfected with ab224892 and two different sgRNAs targeting EZH2. Untransfected cells (WT) and cells transfected with a non-targeting sgRNA (control sgRNA) were used as negative controls. PCR was performed on genomic DNA from the cells with primers flanking the CRISPR targeting site (700 bp amplicon) using a MethylTaq DNA polymerase. The PCR products were tested for CRISPR/Cas9 induced mutations by a T7 Endonuclease I assay. Cleavage at heteroduplex mismatch sites was assessed by agarose gel electrophoresis. Results show that ab224892, when combined with specific sgRNAs provides consistent and effective gene editing.

  • Efficient mutagenesis with ab224892. Zebrafish embryos at the one-cell stage were injected with ab224892 (300 pg) and an sgRNA (30 pg) RNP complex, targeting a gene required for angiogenesis in the brain. The right panel B shows a confocal z-stack of the cranial vasculature of Tg(kdrl:GFP) at 4dpf in dorsal view (anterior to the left). An uninjected control sibling is shown in the left panel A. On average, 85% of the injected embryos display a total absence of hindbrain intracerebral blood vessels at 4dpf. This knock-out causes specific CNS vascular defects showing that the gene of interest is inactivated by the CRISPR/Cas9 system.
     
  • Generation of knock-in mice using ab224892. Fertilized mouse eggs (C57BL/6N) were injected with ab224892 singlestranded
    oligonucleotides, used as a Homology Directed Repair (HDR) template to edit the mouse Smpd3 locus and a guide RNA. The template incorporates a BamHI restriction site for genotyping. Two-cell stage embryos were transferred to a fostermother and 11 neonatal mice were analyzed. Panel A shows a mismatch detection assay using a surveyor assay (Cel1 digest of PCR products). Positive candidates (CEL1-cleaved DNA) are marked with a red *. Panel B shows a BamHI digestion of the PCR products indicating that the donor DNA has been integrated in the genome and specific sequence changes have been introduced. Positive candidates (BamHI-cleaved) are marked with a blue *.

References

ab224892 has not yet been referenced specifically in any publications.

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