Team:Freiburg/parts/favorite parts

From 2013.igem.org

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<p> Our uniCAS Activator  
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<p> Our uniCAS Activator illustrates a fusion protein consisting of dCas9 and VP16 for sequence-specific transactivation of a desired target locus. Virus Protein 16 (VP16) is a transcription factor encoded by the UL48 gene of Herpes simplex virus-1 (HSV-1). Its trans-activation domain is one of the most efficient and we cloned it behind dCas9 in order to activate any gene of interest. For the exact constellation of the associated device take a look at Figure 1.</p>
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                                  <td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/2013/3/30/Freiburg2013_Plasmid_Cas9-VP16.png"> </td>
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                                  <td> <b>Figure 1: Construct design of dCas9-VP16.</b><br>
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dCas9 was linked via a 3 amino acid linker to the 5’ end of the sequence coding for the transactivation domain of VP16. To ensure nuclear localization of the construct, a nuclear localization signal (NLS) was fused to both ends of dCas9-VP16. For detection of expression the fusion protein was tagged with a HA-epitope coding sequence. Its expression was set under control of the CMV promoter and BGH terminator.
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<p>By co-transfecting our RNAimer plasmid which includes the tracrRNA and the separately integrated, desired crRNA, the dCas9 specifically binds to the targeted DNA sequence. With the help of the transactivation domain VP16, transcription factors are recruited and the pre-initiation complex can be built. By placing this construct upstream of a promotor region any gene of interest can be activated.
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dCas9-VP16 was able to induce gene expression up to 10-fold using a single target locus [EMX1] on our tested Reportergen SEAP. Transfecting more than one target locus (multiple targeting) simultaneously, the induction increased in some cases more than two-fold of the additive effect of the tested single targets [EMX1 and T2]. With the multiple targeting we could increase the expression up to 29-fold.
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<td> <b>Figure 5: Results of the SEAP activation with dCas9-VP16 under control of the CMV promoter using different crRNAs.</b><br>
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To quantify the activation properties of dCas9-VP16 the amount of SEAP expression was measured and divided through the expression level of the luciferase Renilla (internal standard). Each sample was measured in biological triplicates.  Bright green columns reflect the negative controls while dark green ones reflect the different samples. The fold induction above each column is related to the basic SEAP expression level of the pRSet (junk DNA) control. T3+4 crRNAs are transcribed from one RNA plasmid while T3 & T4 crRNAs are transcribed from two independent RNA plasmids.
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Revision as of 19:15, 4 October 2013


Our favorite BioBricks

No. 1: BBa_K1150020 - uniCAS Activator

Our uniCAS Activator illustrates a fusion protein consisting of dCas9 and VP16 for sequence-specific transactivation of a desired target locus. Virus Protein 16 (VP16) is a transcription factor encoded by the UL48 gene of Herpes simplex virus-1 (HSV-1). Its trans-activation domain is one of the most efficient and we cloned it behind dCas9 in order to activate any gene of interest. For the exact constellation of the associated device take a look at Figure 1.

Figure 1: Construct design of dCas9-VP16.
dCas9 was linked via a 3 amino acid linker to the 5’ end of the sequence coding for the transactivation domain of VP16. To ensure nuclear localization of the construct, a nuclear localization signal (NLS) was fused to both ends of dCas9-VP16. For detection of expression the fusion protein was tagged with a HA-epitope coding sequence. Its expression was set under control of the CMV promoter and BGH terminator.

By co-transfecting our RNAimer plasmid which includes the tracrRNA and the separately integrated, desired crRNA, the dCas9 specifically binds to the targeted DNA sequence. With the help of the transactivation domain VP16, transcription factors are recruited and the pre-initiation complex can be built. By placing this construct upstream of a promotor region any gene of interest can be activated. dCas9-VP16 was able to induce gene expression up to 10-fold using a single target locus [EMX1] on our tested Reportergen SEAP. Transfecting more than one target locus (multiple targeting) simultaneously, the induction increased in some cases more than two-fold of the additive effect of the tested single targets [EMX1 and T2]. With the multiple targeting we could increase the expression up to 29-fold.

Figure 5: Results of the SEAP activation with dCas9-VP16 under control of the CMV promoter using different crRNAs.
To quantify the activation properties of dCas9-VP16 the amount of SEAP expression was measured and divided through the expression level of the luciferase Renilla (internal standard). Each sample was measured in biological triplicates. Bright green columns reflect the negative controls while dark green ones reflect the different samples. The fold induction above each column is related to the basic SEAP expression level of the pRSet (junk DNA) control. T3+4 crRNAs are transcribed from one RNA plasmid while T3 & T4 crRNAs are transcribed from two independent RNA plasmids.

No. 2: BBa_K1150024 - uniCAS Histone Modifier

Figure X: CMV:dCas9-G9a (BBa_K1150024)
dCas9 was fused via a 3 amino acid linker to G9a. The resulting fusion protein was flanked by NLS sequences and tagged by a HA epitope. The CMV promoter and BGH terminator were chosen to control gene expression.


This device combines the dCas9 protein with the SET-domain of the murine histone methyltransferase G9a. dCas9 enables not only sequence specific, but also multiple targeting of any requested DNA sequence. Hence, coupling of dCas9 to the effector G9a allows for specific methylation of histone (Figure XXXX).
Such methylations are a hallmark of gene repression. One interesting fact about histone modification is the capability to spread the activity state over the surrounding chromatin via reader proteins. So the information of e.g. "repressed state" can, once specifically introduced, be propagated over a whole locus.

We included the G9a histone methylase to the uniCAS toolkit for specific histone methylation. With the device BBa_K1150024, consisting of dCas9 and G9a, we were able to decrease the endogenous VEGF expression in HEK293T cells about 50%, depending on the locus targeted (Figure XXXXXX).

Chromatin remodeling, resulting in repression of endogenous genes, is possible by fusing the histone methyltransferase G9a to dCas9.
dCas9-G9a is our most efficient repressiv device!

Figure X: Endogenous, stable repression by dCas9-G9a
HEK293T cells have been trancfected with the BBa_K1150024 device and the RNAimer plasmid containing the different crRNA target sites for the endogenous VEGF locus. 12 hours after transfection the medium was change and 24 hours after medium change we harvested the supernatant and performed VEGF measurments by ELISA. As a control that the repressive effect of our proteins is not based on the sterical block of the transcription, we tested against the catalytic inactive dCas9. So every detectable effect is due to the G9a targeted to this locus.(n=3, p<0.05 is marked by asterisks)

No. 3: BBa_K1150034 - uniCAS RNAimer

Figure XXXXX: RNAimer (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, H1 and U6. The combination of multiple crRNAs can be easily done by digestion with enzymes of prefix and suffix and ligation of these parts and the RNAimer backbone according to iGEM standard assembly.

As dCas9 requires special RNAs for binding to the DNA, we designed a RNA plasmid containing the structure giving tracrRNA and the DNA binding crRNA. The crRNA is responsible for sequence specific DNA-binding of the entire RNA-protein complex.
The crRNA can be easily introduced to the plasmid by digesting the backbone with BbsI and insertation of an double stranded oligo. 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.

We could show that a RNAimer plasmid containing two crRNAs is as effective as co-transfected crRNAs (Figure XXXX). Besides the RNAimer enables multiple targeting, which means to use several crRNAs for one requested locus. Even for multiple targeting different RNAimers can be combined using the iGEM BioBrick system. And as the results show, multiple targeting is more effective concerning the influence the effector has on gene expression (Figure XXXXX).

Figure XXXXXX: RNAimer in comparison to two RNA plasmids
HEK-293T cells were transfected with CMV:dCas9-VP16 ad either two RNA palsimd (left) or one RNAimer plasmid (right), containing the same crRNAs (T3, T4). The suppernant was taken and SEAP activity measured. The bars represent the mean of biological triplicates with standard deviation.
Figure 7: Effect of multiple targenting using the RNAimer plasmid
Mamalian HEK-293T cells were transfected with CMV:dCas9-VP16 and different crRNA targets for the same locus. After 48h the suppernant was taken for SEAP measurement. The bars represent the mean of biological triplicates with standard deviation. The last bar shows the activation of the SEAP gene while two differnt crRNAs target the CMV:dCas9-VP-16 construct to the same locus.

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