Team:Freiburg/Project/toolkit

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The uniCAS toolkit - Customize your experiments!

You want to have a maximum of activation or repression of your genes by a minimal effort? Then you have to use the uniCAS toolkit provided by the iGEM team Freiburg 2013. All you have to do is:

Click yourself through the routine below
Order the appropriate plasmids and oligos
Conduct a minimal of cloning
Start your personalized experiment

By the end of the routine you will get a personal manual. All you need to use the uniCAS toolkit will be described there. Best of all: The uniCAS toolkit is all open source!

Activation

Repression

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)

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