Team:Freiburg/Project/crrna

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:

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.

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:

1. Search at your desired target sequence for a NGG (the PAM sequence) at the non-coding strand.
2. Extract the 30 nucleotides before (5') the PAM sequence.
3. Extract the reverse complement.
4. Add TAAAAC at the 5' end. This will be your second oligo.
5. 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 )

Technical Information

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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 an 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 crRNAs on one plasmid.

As it is important that the RNAs are not being marked for protein expression the RNA polymerase III is required for transcription. RNA polymerase III mainly synthesize small non-coding RNAs (e.g. tRNAs or rRNAs) whereas the commonly 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 exclusively 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 at once

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. 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 fluorescent proteins The fluorescence intensity 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

As we wanted to improve the efficiency of our gene regulation tool kit, we tried to target several loci upstream of the reporter gene's promoter at once. Thus, we ordered crRNAs that are complementary to sequences on the SEAP reporter plasmid with different distances to the promoter.

TABELLE: Targets on SEAP reporter plasmid

HEK cells were transfected with the SEAP reporter plasmid, Cas9-VP16 (iGEM standard), one or two RNA plasmids and a plasmid coding for Renilla luciferase for a internal standard (to eliminate variabilities of different cell numbers or expression levels). The total amount of RNA plasmids was always the same, so it can be exclude that an increase of SEAP expression is due to more available crRNA. When combining the Targets EMX1 and T2 there could be observed a higher SEAP activation than the sum of the single targets (Fig. 5).

Fig. 5: Effects of different target numbers SEAP activity was divided through luminescence intensity of Renilla luciferase. The bars represent the mean of biological triplicates with standard deviation. All samples were transfected with CMV::Cas9-VP16 in pSB1C3 and with no (left), one (middle) or two RNA plasmids (right).

RNAimer - the multiple RNA plasmid

In order to reduce the amount of plasmids for transfection when intending to target several genes or target sites at once, we wanted to join the required crRNAs on one RNA plasmid. This was easily manageable by using the iGEM standard assembly: We inserted the oligos seperately into BBa_K1150034 by digesting with BbsI and ligation. Afterwards the whole inserts can be combined by using the restriction enzymes of the prefix and suffix.

FIGURE: RNAimer (mit Verweis!)

When compared to two RNA plasmids containing the same crRNAs the RNAimer causes the same SEAP activation in HEK cells transfected with this plasmid(s), CMV::Cas9-VP16, Renilla luciferase and the SEAP reporter plasmid (Fig. 7).

Figure 7: RNAimer in comparison to two RNA plasmids SEAP activity was divided through luminescence intensity of Renilla luciferase. The bars represent the mean of biological triplicates with standard deviation. All samples were transfected with CMV::Cas9-VP16 in pSB1C3 and with two (left) or one RNA plasmid (right), each time coding for the same crRNAs. T3+4: RNAimer with T3 and T4 crRNA; T3 & T4 two different RNA plasmids with T3 and T4 crRNA.

Summary

We have shown, that the efficiency of transcription activation can be enhanced by targeting different loci upstream of a promoter simultaneously. While Cheng et al. [3] yielded an up to 8 fold activation by using at least three different targets in comparison to the highest activation of a single target, we were able to achieve a three fold increase by using only two different targets.
With our toolkit it is possible to induce a gene regulation by transfecting only two plasmids (Cas9 with effector and RNAimer) that can be combinied with no effort in accordance to the experimental setup.
For future application researchers may be able to render gene expression by transfecting even only plasmid containing the crRNA: when Cas9 will be stable integrated into the genome of cells or model organisms.

About the ability of activating multiple genes we could not make a statement as the activation of our reporter plasmids could not be detected at all with Cas9-VP16. This may be due to the high background expression of CMV_min.

References