Team:Freiburg/Project/crrna

Targeting

In our uniCAS Toolkit we engineered the CRISPR/Cas9 system for future applications to regulate gene expression. The key components for the binding of the catalytically inactive Cas9 (dCas9) to its targets are two small, non-coding RNAs: the CRISPR-RNA (crRNA) and the tracrRNA. These RNAs that guide our dCas9 to specific DNA sequences have to be cotransfected with the dCas9-plasmids. Therefore we designed an RNA plasmid, termed RNAimer , which contains all required RNAs for efficiently guiding the dCas9 protein to the required DNA - even if multiple DNA sites should be targeted.

As an essential part of our toolkit is the binding of our protein to DNA, we evaluated this in detail: Various DNA target sequences were compared to evaluate the best guiding crRNAs. Evaluated parameters were varying loci and GC-content. Based on these results, we tested if dCas9 isd able to bind to multiple targets .

In order to simplify the search for potential target sequences, we programmed the crRNA design tool and provide it to the iGEM community. As the binding of the RNA-guided Cas9 requires a protospacer adjacent motif (PAM), the tool determines all possible regions of the desired DNA sequence, taht is feeded into the tool. Eventually, you obtain the position of your target sites and the sequences of the oligos that have to be inserted into the RNAimer - ready to order!

Introduction

As dCas9 requires two small RNAs to facilitate the binding to the DNA, we designed an RNA plasmid containing the tracrRNA, where the crRNA can be introduced easily by digesting with BbsI and inserting oligos, that have been annealed previously. 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 and nuclear export 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] . 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: Oligos are inserted the separately 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 6).

Figure 6: Assembly and function of the RNAimer For each desired crRNA, one 'RNA plasmid' (=containing the crRNA after ligation) has to be opened by digestion with BbsI. Afterwards the annealed crRNA oligos will be ligated. After that, the different RNA inserts can be assembled using the idempotent iGEM cloning strategy. So three different crRNAs will be transcribed.

Results

To test this strategy functionally we performed SEAP (Secreted Embryonic Alkaline Phosphatase) assays with dCas9-VP16. HEK-293T cells were transfected with dCas9-VP16 and different RNAimer plasmids, containing different crRNAs. Either we transfected two crRNAs on different plasmids or both sites on a single plasmid. This led to the same activation of SEAP expression (Figure 7).

Figure 7: SEAP activation with dCas9-VP16 and RNAimer. Activation by dCas9-VP16 with two targets at once. In the case of T3+4 the loci were on the same plasmid, whereas in the case of T3&T4 two separate plasmids were transfected. Both combinations display a strong activation of the CMVmin promoter, driving the SEAP promoter. The activation is comparable. This is a hint, that there is no decrease in efficiency when combining several loci on one plasmid. HEK -293T cells were seeded at 65,000 cells in 24-well format. After 24 hours cells were transfected following standard procedure. After 48 hrs of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.

Discussion

We have shown that our RNA plasmid, the RNAimer, works as expected. The combination of two targets does not seem to decrease the efficiency of targeting as the activation is comparable if transfecting two separate plasmids or combining both targets in one plasmid. With our toolkit it is possible to induce a gene by transfecting only two plasmids (dCas9 with effector and RNAimer) that can be recombined with no effort in accordance to the experimental setup.
For future application researchers will be able to alter gene expression by transfecting even only plasmids containing the crRNA: when dCas9 will be stable integrated into the genome of cells or model organisms, as recently done by Gilbert et al. [4].

References

(1) Dieci G,et al. (2007). The expanding RNA polymerase III transcriptome. Trends Genet. 2007 Dec;23(12):614-22.

(2) Myslinski E, et al. (2001). 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.

(3) Cheng AW, et al. (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013 Aug 27. doi: 10.1038/cr.2013.122.

(4) Gilbert LA,et al. (2013). CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell. 2013 Jul 18;154 (2):442-51.

Introduction

One of the biggest advantages of the CRISPR/Cas9 system compared to other transcription activators (e.g. Zn fingers, TALEs) is that only one protein is required for targeting several DNA loci: For a new target just another crRNA has to be expressed. So a RNA plasmid was designed containing the tracrRNA, where the crRNA can be easily introduced.
With this RNA plasmid and another plasmid containing the dCas9-effector fusion protein it is possible to target several DNA sites at once by transfecting only two plasmids. This allows the simultaneous regulation of different genes or a stricter control of one gene by targeting several loci of this gene the same time.

But at first we had to evaluate the adequate DNA sequences for targeting, because the results of our first experiments we recognized that different target loci cause different effects. Thus, we cloned 5 different target sites 26 bp upstream of the CMV_min promoter of a SEAP reporter plasmid. Additionally we designed crRNAs in various distances upstream of this promoter (Tab. 1). This target sites were evaluated by activating SEAP expression with dCas9-VP16.

Results

Evaluation of different target sequences

At first we tested different target sequences at the same distance from the promoter. To do so we had to insert the target sequences into the SEAP reporter plasmid in front of the CMV_min.
HEK-293T cells were transfected with one of these SEAP plasmids and a plasmid containing dCas9-VP16, the tracrRNA and the appropriate crRNA. The results show different activation efficiencies as it can be seen in Figure 8: from no activation at all (VEGF VZ -573 and +434) to a 5 fold increase of SEAP activation. With the tested target sequences that have a GC content of 70 % there was no activation, whereas it was possible to induce SEAP expression with the target sequences with a GC content of about 60 % (compare Tab. 1).

Figure 8: Targets with different sequences The grey bars represent the SEAP activity of HEK-293T cells transfected with dCas9-VP16 but no crRNA normalized to one. The blue bars show fold induction of SEAP activity by tansfecting dCas9-VP16 with crRNAs in comparison to the appropriate control without crRNA. An activation is visible for nearly all targets, but the fold-induction is differing strongly. All values are means of three biological replicates. Error bars represent standard deviation. HEK -293T cells were seeded at 65,000 cells in 24-well format. After 24 hours cells were transfected following standard procedure. After 48 hrs of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.

Secondly, we evaluated different distances upstream of the promoter. Thus, we targeted 4 different loci on the reporter plasmid (EMX1, T2, T3 & T4; see Tab. 1).
HEK-293T cells were transfected with a CMV_min:SEAP plasmid that contains EMX1, CMV:dCas9-VP16 and the RNA plasmid. The results show a very high increase in SEAP expression when EMX1, which is the closest target site to the promoter, is targeted (Fig. 2). Activation becomes weaker, the lager the distance between promoter and target sites gets(compare Tab. 1).

Figure 9: Targets with different distances to the promoter Activation of CMVmin:SEAP via dCas9-VP16 with different distances to the promoter in HEK-293T cells. The grey bar represents the SEAP level without crRNA, the blue bars were activated by different crRNAs in increasing distance to the promoter (EMX1 the closest, T4 with the greatest distance. A clear decrease in activation is observed with increasing distance from promoter. All epxeriments were performed in biological triplicates. Error bars represent standard deviation. HEK-293T cells were seeded at 65,000 cells in 24-well format and 24 hours later transfected following the standard procedure. After 48 h of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.

Activation of different genes at once

In order to test the simultaneous activation of several genes we assembled three plasmids containing different fluorescent proteins (Figure 10). 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 less interference of the emitted light. These plasmids were transfected into HeLa cells with corresponding RNA plasmids and were checked for fluorescence which is depicted in Figure 11.

HEK cells were transfected with different combinations of these plasmids and dCas9-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. As it can be seen in figure 12 there is no activation observable for dCas9-VP16.

Fig. 12: 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 plasmids 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 dCas9-VP16.

Unfortunately dCas9-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 dCas9-VP16).

Stricter gene regulation by targeting different loci simultaneously

In order to improve the efficiency of our gene regulation toolkit, we tried to target several loci upstream of the promoter of the reporter gene at once. Thus, we designed crRNAs that are complementary to sequences on the SEAP reporter plasmid with different distances to the promoter (Tab. 1).

HEK-293T cells were transfected with the CMV_min:SEAP reporter plasmid, dCas9-VP16 (iGEM standard), one or two RNA plasmids and a plasmid coding for Renilla luciferase for an internal standard (to eliminate variabilities of different cell numbers or expression levels). The total amount of RNA plasmids is kept constant, so an increase of SEAP expression due to more available crRNA can be excluded. By combining the targets EMX1 and T2 a higher SEAP activity than the sum of the single targets can be observed(Figure 13).

Figure 13: Effects of different target numbers Activation of CMVmin:SEAP was evaluated by the usage of single targets and combined targets. Every target itself displays an activation of the minimal promoter. By combining two targets (EMX1 & T2), an even stronger activation is visible, that exceeds the additive effect of single plasmids. HEK-293T cells were seeded at 65,000 cells in 24-well format. After 24 hours cells were transfected following standard procedure. After 48 h of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.

Discussion

From the few target sites we evaluated we could draw the conclusion that a target should be chosen like this:

• The distance to the promoter should be kept at minimum.
• Target sites with a GC content around 60% should be preferred.

As we tested our target sequences with dCas9-VP16, stricly speaking, we can only make a statement about the best target sites for gene activation.
Moreover there may be other parameters that influence the effects of dCas9-VP16 on SEAP (e.g. the secondary structure of the crRNA). But for the distance to the promoter Mali et al. [1] came to the same conclusion: Only the target site that had the shortest distance to the promoter showed a high increase of activation.

By targeting different loci upstream of a promoter simultaneously the efficiency of transcription activation can be enhanced. While Cheng et al. [2] yielded an activation up to 8-fold 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.
About the ability of activating multiple genes we could not draw a conclusion as the activation of our reporter plasmids could not be detected at all with dCas9-VP16. This may be due to the high background expression of CMV_min. Nevertheless Cheng et al. showed that it is possible to target up to three genes at once [2].

References

(1) Mali P,et al. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013 Sep;31(9):833-8.

(2) Cheng AW, et al. (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013 Aug 27. doi: 10.1038/cr.2013.122.