Diagenode

Cas9 Nuclease protein NLS

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Catalog Number
Format
Price
C29010001
75 µg/ 25 µl
$260.00

Use highly purified Cas9 Nuclease protein NLS for efficient targeted double-stranded DNA cleavage. The N-terminal and C-terminal Nuclear Localization Signal (NLS) of the protein enable rapid nucleus delivery. Cas9 Nuclease protein NLS and guide RNA (user-supplied) form a complex that specifically cleaves double-stranded DNA.

  • Fewer off-target cleavage events vector-based systems
  • High purity: >95%
  • High concentration (3µg/µl)
  • Low endotoxin level
  • DNA-free assay
  • Reduced incidence of clogging in micro-injections
  • High activity confirmed by in vitro and in vivo assays

Protocols

  • Quality control

    In vitro DNA

    Figure 1. In vitro DNA cleavage assay using the Diagenode Cas9 Nuclease protein NLS
    A Cas9 reporter plasmid linearized with PvuI was incubated with (lanes 2 and 3) or without (lane 1) the Diagenode Cas9 NLS protein (Cat. No. C29010001) 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

    Figure 2. In vitro transfection assay using the Diagenode Cas9 Nuclease protein NLS
    HEK293T cells were transfected with the Diagenode Cas9 protein NLS (Cat. No. C29010001) and two different sgRNAs targeting EZH2. Untransfected cells (WT) and cells transfected with the Cas9 nuclease and 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 the MethylTaq DNA polymerase (C09010010). 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. These results show that the Cas9 Nuclease Protein NLS, when combined with specific sgRNAs provides consistent and effective gene editing.

    Efficient mutagenesis

    Figure 3. Efficient mutagenesis with the Diagenode Cas9 Nuclease protein NLS
    Zebrafish embryos at the one-cell stage were injected with the Diagenode Cas9 Nuclease protein NLS (Cat. No. C29010001, 300pg) and an sgRNA (30pg) RNP complex, targeting a gene required for angiogenesis in the brain. Figure 3B 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 figure 3A. 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.

    Efficient mutagenesis

    Figure 4. Generation of knock-in mice using the Diagenode Cas9 Nuclease Protein NLS
    Fertilized mouse eggs (C57BL/6N) were injected with the Diagenode Cas9 Nuclease protein NLS (Cat. No. C29010001), single- stranded 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 foster- mother and 11 neonatal mice were analyzed. Figure 4A shows a mismatch detection assay using a surveyor assay (Cel1 digest of PCR products). Positive candidates (CEL1-cleaved DNA) are marked with a red *. Figure 4B shows a BamHI digestion of the PCR products indicating that the donor DNA has been integrated in the genome and speci c sequence changes have been introduced. Positive candidates (BamHI-cleaved) are marked with a blue *. More details are shown in the table below.

    Material Final concentration Injected sites Number od embryos injected Number of embryos transferred Number of neonates KOs Kls
    Cas9 Nuclease 30 ng/μl Cytoplasm
    +
    Pronucleus
    123 114 11 6/11
    (54.5 %)
    6/11
    (54.5 %)

    sgRNA

    50 ng/μl

    ssODN

    1000 ng/μl
  •  Protocols
    CRISPR/Cas9 editing: mutation detection with mismatch cleavage assay
    Genome editing using CRISPR/Cas9 is used for targeted mutagenesis. But because genome editing doe...
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    Transfection of CRISPR/Cas9 Nuclease NLS ribonucleoprotein (RNP) into adherent mammalian cells using Lipofectamine® RNAiMAX
    The CRISPR/Cas genome editing system consists of a single guide RNA (sgRNA) and the Cas9 endonucl...
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  •  Documents
    Cas9 Nuclease protein NLS DATASHEET
    Full length recombinant Streptococcus pyogenes Cas9 Nuclease, with an N-terminal and C-terminal N...
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    Accurate QC to optimize CRISPR/Cas9 genome editing specificity POSTER
    The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease res...
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  •  Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: Cas9 Nuclease protein NLS (Diagenode Cat# C29010001). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    zHSF1 modulates zper2 expression in zebrafish embryos
    Mennetrier L. et al.
    HSF1 is a transcription factor that plays a key role in circadian resetting by temperature. We have used zebrafish embryos to decipher the roles of zHsf1, heat and light on zper2 transcription in vivo. Our results show that heat shock (HS) stimulated zper2 expression in the dark but has no cumulative effect combined...

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    Sep 19-Sep 20, 2018
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    Sep 21-Sep 22, 2018
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