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NLS-Cas9-EGFP
NLS‐Cas9‐EGFP-NLS is a fusion protein containing a nuclear localization sequence (NLS) on its N terminal and EGFP-NLS on the C terminal.
Data sheet
| Storage | -70°C. Avoid freeze thaw cycles. |
| Background | NLS‐Cas9‐EGFP-NLS is a fusion protein containing a nuclear localization sequence (NLS) on its N terminal and EGFP-NLS on the C terminal. The Cas9 RNP complex can localize to the nucleus immediately upon entering the cell. The EGFP can be taken as a reporter for tracking or sorting transfected cells, which creates the possibility of enriching cell populations for desired genome edits via fluorescence activated cell sorting (FACS). |
| Formulation | Recombinant NLS-Cas9-EGFP-NLS 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. |
| Concentration | 50 μg/ 50 μl or 1 μg/μl or molar concentration 5.31 µM. |
More info
For molar concentration and other related product information, please refer to:
In vitro Cas9 mediated digestion of DNA
1. Add the following components to a sterile, nuclease-free tube on ice:
|
Components |
Volume |
Final Concentration |
|
|
sgRNA (300 nM) |
2 µl |
~30 nM |
|
|
Cas9-EGFP 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.
3. Analyze fragments by running an agarose gel electrophoresis, or do transfection for target gene tracking.
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. PNAS2015 vol. 112 | no. 38 p11870-11875
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