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Data sheet
| Background | The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea. CRISPR uses a Cas9 protein to recognize DNA sequences, with target specificity solely determined by a small guide (sg) RNA and a protospacer adjacent motif (PAM) Upon binding to target DNA, the Cas9-sgRNA complex generates a DNA double-stranded break. Based on this RNA- guided nuclease activity, CRISPR has been showed to be a powerful tool in editing the genomes of a broad range of organisms. Furthermore, a repurposed, nuclease-deactivated Cas9 (dCas9) protein has been used to regulate endogenous gene expression and labeling of genomic loci in living and fixed cells. |
| Formulation | Recombinant dCas9 (D10A & H840A) protein expressed in E. coli supplied in a buffer of 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 300 mM NaCl, and 50% (v/v) Glycerol. |
| Host/Source | E. coli |
| Applications | Recombinant dCas9 (D10A & H840A) protein is suitable for use in imaging of genomic oci in living cells and fixed cells as well as for gene expression regulation. |
| Stability/Storage | Recombinant dCas9 (D10A & H840A) protein in solution is temperature sensitive and must been stored at -20°C or below to prevent degradation. Avoid repeated freeze /thaw cycles and keep on ice when not in storage. Stable for 1 year from the date of shipping when stored and handled properly. |
| Concentration | 1 μg/μl, molar concentration 6.26 μM. |
| Physical appearance | Sterile filtered colorless solution. |
More info
For molar concentration and other related product information, please refer to:
CRISPR/Cas9 related enzyme proteins, antibodies, and other reagents
RNP Complex Formation and Target DNA binding
1. Add the following components to a sterile, nuclease-free tube on ice:
|
Components |
Volume |
Final Concentration |
|
|
sgRNA (300 nM) |
2 µl |
~30 nM |
|
|
dCas9 Nuclease Protein (1000 nM) |
0.60 µl |
~30 nM |
|
|
10X Cas9 Nuclease Reaction Buffer |
2 μl |
1X |
|
|
Nuclease-free H2O |
12.4 μl |
- |
|
|
Pre-Incubate for 15 minutes at room temperature |
|||
|
Substrate DNA (30 nM) |
3 μl |
3 nM |
|
|
Total Volume |
20 μl |
- |
|
2. Collect all components by a brief centrifugation. Incubate the reaction at 37 °C for 1 hour.
References:
1. Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA Amin Aalipour et al. Clinical Chemistry 2018 Vol. 64, Issue 2 p307-p916
2. Purified Cas9 Fusion Proteins for Advanced Genome Manipulation Jovan Mircetic et al. Small Methods 2017 1, 1600052
3. Disruptive non-disruptive applications of CRISPR/Cas9 Jonathan LSchmid-Burgk Current Opinion in Biotechnology December 2017, Volume 48 Pages 203-209
4. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins Toshitsugu Fujita et al. Genes to Cells Volume 21, Issue 4 April 2016 Pages 370–377
5. High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding Evan A Boyle et al. PNAS 2017 May, 114 (21) 5461-5466
6.CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells Wulan Deng et al. PNAS 2015 vol. 112 | no. 38 p11870-11875
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