You have chosen to do a gene activation experiment using a non inducible Cas9-VP16 device. Therefor you first have to order the following plasmids from the iGEM parts registry :
| No. |
Biobrick |
Device |
Order |
GeneBank File |
| 1 |
BBa_K1150017 |
CMV-HA-NLS-Cas9-NLS-BGH |
order |
genebank |
| 2 |
BBa_K1150020 |
CMV-HA-NLS-Cas9-L3-VP16-NLS-BGH |
order |
genebank |
| 3 |
BBa_K1150034 |
crRNA - plasmid |
order |
genebank |
Note:
All plasmids are optimised for an expression in mammalian systems. The devices are also available containing an SV40 instead of an CMV promotor (have a look at our parts side). This system was tested mainly in CHO, HEK-293-T and HeLa cells.
Design of the crRNA plasmid:
Use our crRNA design tool to design the crRNAs that are needed to target your gene of interest. It will print out possible target sides and the appropriate oligos. Order the oligos by the company of your choise. We recommend to order multiple crRNA oligos for the targeting of one locus.
- Oligo anneal the ordered oligos to gain the the desired crRNAs
- Digest BBa_K1150034 with Bbs1
- Ligate crRNAs (step 1) into pre Bbs1 cut backbone. Do so for all designed crRNAs.
Now that you have created the desired crRNA plasmids it is possible to fuse all crRNA loci together into one crRNA plasmid (recommended). If you want to use them individually just skip this part.
Experimental design (recommendation for a mammalian cell culture):
- Transfect the appropriate crRNA - plasmid togehter with BBa_K1150017 that has no effector for control
- Transfect BBa_K1150020 without any crRNA plasmid as off target control
- Transfect BBa_K1150020 with all desired crRNA plasmids (seperate and in combinitions)
"
