Team:Freiburg/Project/crrna
From 2013.igem.org
Line 378: | Line 378: | ||
- | <p style="padding-top:10px"> <i>(designed analog to: Cong L, Ran FA, Cox D, Lin S, Barretto R,Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex (2013 Jan 3). Genome Engineering using CRISPR/Cas Systems. Science. DOI: 10.1126/science.1231143 )</i><br> | + | <p style="padding-top:10px"> <i>(the oligos are designed analog to: Cong L, Ran FA, Cox D, Lin S, Barretto R,Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex (2013 Jan 3). Genome Engineering using CRISPR/Cas Systems. Science. DOI: 10.1126/science.1231143 )</i><br> |
</p> | </p> | ||
Revision as of 16:43, 29 September 2013
Targeting
crRNA design tool
This tool helps you to design a crRNA-insert for dCAS RNA plasmid: "uniCAS RNaimer" ( BBa_K1150034 ).
Using this tool you do not have to do this on your own. Just insert the desired target sequence and you get all different oligo possibilities and their positions. The oligos contain overhangs which fit to this plasmid's BbsI-overhangs and are ready to use.
The two different target possibilities are the coding and non-coding strand, depending on the desired target sequence.
a) 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.
b) For repression of gene transcription by targeting the non-coding sequence it´s crucial to target the template DNA strand.
Therefore the oligos must be designed as follows:
- Search at your desired target sequence for a NGG (the PAM sequence) at the non-coding strand.
- Extract the 30 nucleotides before (5') the PAM sequence.
- Extract the reverse complement.
- Add TAAAAC at the 5' end. This will be your second oligo.
- Take the sequence from step 2 and add AAAC at the 5' end and GT at the 3' end. This will be your first oligo.
(the oligos are designed analog to: Cong L, Ran FA, Cox D, Lin S, Barretto R,Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex (2013 Jan 3). Genome Engineering using CRISPR/Cas Systems. Science. DOI: 10.1126/science.1231143 )
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.
Fig. 1: RNA plasmid (BBa_K1150034) |
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. Fig. 2: Plasmids encoding the fluorescent proteins Each fluorescent protein is driven by a CMV minimal promoter, that can be switched on by binding of TetR-VP16 to the TetO sequence. Between TetO and CMVmin there is a target site for Cas9 binding, a different on each plasmid. The flourescent were fused to signal sequences for subcellular localization, so mCherry will be in the nucleus, GFP in the Golgi apparatus and BFP at the membrane. T: terminator. |
Fig. 3: Microscopy pictures of fluorescent proteins expressed in HeLa cells Fluorescence pictures were taken of fixed HeLa cells transfected with Golgi-GFP and mCherry-NLS. Channels of GFP and mCherry were merged. All pictures have the same scale. |
Fig. 4: Activation of expression of different fluorescet proteins The fluorescence intenstity of each cell was analysed by flow cytometry. The mean fluorescence intensity was calculated with the intensities of the cells which were brighter than untransfected cells. The bars represent the mean with standard deviation of these mean fluorescences of three different cell populations. blue: only the plasmid containing the fluorescent proteins with minimal promoter were transfected; green: the minimal promoter driven fluorescent proteins were cotransfected with TetR-VP16; yellow: the minimal promoter driven fluorescent proteins were cotransfected with Cas9-VP16. |
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