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Team:Freiburg/Project/crrna
From 2013.igem.org
Targeting
crRNA design tool
This tool helps you to design a crRNA-insert for pX334a. The oligos contain overhangs which fit to the BbsI-overhangs by pX334a.
For repression of gene transcription by targeting the coding sequence
it´s crucial to target the non template (= coding) DNA strand.
Therefore the oligos must be designed as
follows:
- Search at your desired target sequence for a CCN (reverse complement of the PAM sequence) at the coding strand.
- Extract the following (3') 30 nucleotides.
- Extract the reverse complement.
- Add AAAC at the 5' end and GT at the 3' end. This will be your fist oligo.
- Take the sequence from step 2 and add TAAAAC at the 5' end. This will be your second oligo.
Multiple targeting
Introduction
One of the biggest advantages of the CRISPR-Cas system compared to other transcription activators (e.g. Zn fingers, TALEs) is that only one protein is required for targeting several DNA sites: For a new target there has to be just another crRNA. We designed a RNA plasmid containing the tracrRNA, where the crRNA can be introduced easily by digesting with BbsI and inserting two previous annealed oligos. Two of these RNA plasmids (with different crRNAs) can be fused using the iGEM biobrick system. This way it is possible to get two or more crRNA on one plasmid.FIGURE 1
As it is improtant that the RNA are not being marked for protein expression the RNA polymerase III is required for transcription. RNA polymerase III mainly synthesis small non coding RNAs (e.g. tRNAs or rRNAs) whereas the commly used polymerase II is responsible for transcription of mainly mRNAs [1,2] . We chose the human U6- and H1-promoter to drive the RNAs as they are exclusivle recognized by polymerase III [2] .
With this RNA plasmid and another plasmid containing the Cas9-effector fusion protein it is possible to target several DNA sites at once by transfecting only two plasmids. This could mean the simultaneous regulation of different genes or a stricter controlling of one gen by bringing more effector domains to this gene.
Results
Activation of different genes
In order to test the simultaneously activation of several genes we assembled 3 plasmids containing different fluorescent proteins. Every protein is fused to a different signal for intracellular localization. Thus, we were able to distinguish better between the different fluorescent proteins, because there will be no interference of the emitted light.FIGURE 2
HEK cells were transfected with different combinations of these plasmids and Cas9-VP16 or TetR-VP16 (4 fold amount of effector DNA) After 2 days of expression the cells were analysed by flow cytometry. This way the fluorescence intensity of every cell could be determined (Fig. 3).
FIGURE 3
Unfortunately Cas9-VP16 was not able to increase the intensity of any fluorescent protein at all, even when transfected exclucively, whereas TetR-VP16 strongly activates the expression of fluorescent proteins. This may be due to the higher number of binding sites for TetR (16 in comparison with 1 for Cas9).
Stricter gen regulation by targeting different loci simultaneously
multiple RNA plasmid
Summary
So it is not possible to make a statement about the ability of activating multiple genes.References
[1] Dieci G,et al. (1987). The expanding RNA polymerase III transcriptome.Trends Genet. 2007 Dec;23(12):614-22.
[2] Myslinski E, et al. (1987). An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene.Nucleic Acids Res. 2001 Jun 15;29(12):2502-9.
