Team:Freiburg/Project/crrna

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CrispR

Targeting

crRNA design tool

Enter Target Sequence

Choose Program

Which strand should be targeted?

non coding strand coding strand

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:

  1. Search at your desired target sequence for a CCN (reverse complement of the PAM sequence) at the coding strand.
  2. Extract the following (3') 30 nucleotides.
  3. Extract the reverse complement.
  4. Add AAAC at the 5' end and GT at the 3' end. This will be your fist oligo.
  5. Take the sequence from step 2 and add TAAAAC at the 5' end. This will be your second oligo.

CONTENT

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)
Our RNA plasmid contains the tracrRNA and a site where the desired crRNA can be inserted. Both RNAs are driven by different human RNA polymerase III promoters. Assembly of multiple crRNAs can be easily done by digestion with enzymes of prefix and suffix and combining these parts according to iGEM standard assembly.

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.
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. 4).
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.
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.

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