In vitro CRISPR/Cas9 reaction for the digestion of a gRNA target sequence:
1) Set up the reaction mixture as below.
| Components | Amount | Range of Amount |
|---|---|---|
| Cas9 protein | 500 ng | 100 - 1000 ng |
| gRNA | 350 np | 70 - 700 ng |
| Target DNA | 250 - 1000 ng | |
| 10x NEB buffer3 | 2 µl | |
| ScaI restriction enzyme | 1 µl | |
| 1 mg/ml BSA | 2 µl | |
| H20 | 20 µl |
2) Incubate the reaction for 1-3 hours at 37°C.
3) Add 2μl of 10x DNA loading to the reaction and run on 1% agarose gel.
Microinjection mixture for one-cell embryos of mouse or drosophila/zebrafish
1) Set up the mixture as below.
| Mouse | Concentration (ng/µl) | Range of Concentration (ng/µl) |
|---|---|---|
| Cas9 Protein | 40 | 10-200 |
| gRNA | 16 | 4-80 |
| Donor DNA | 32 | 5-100 |
| Drosophilia/ Zebrafish | Concentration (ng/µl) | Range of Concentration (ng/µl) |
|---|---|---|
| Cas9 Protein | 500 | 100-2000 |
| gRNA | 200 | 40-8000 |
| Donor DNA | 400 | 40-8000 |
2) Incubate for 15 minutes at 37°C.
3) Inject 1μl of the mixture into the one-cell embryos of a mouse or a drosophila/zebrafish
Trending Articles:
- Correction of a pathogenic gene mutation in human embryos. Ma, H. et al. Nature 2017
- Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. The Doudna lab and Marson lab, PNAS. 2015
- Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Liang, et al. J. Biotech. 2015
- Efficient intracellular delivery of native proteins. D’Astolfo, et al. Cell 2015
- DNA-free genome editing in plants with preassembled CRISPR-CAS9 ribounucleoproteins. Woo, et al. Nature Biotech. 2015