Team:Freiburg/Highlights/2

From 2013.igem.org

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<p id="h4"><font size="5"><i style="margin-left:95px;">In the last months we were able to ...</i></font></p>
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<p id="h4"><font size="5"><i style="margin-left:85px;">In the last months we were able to ...</i></font></p>
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     <li> ... construct a catalytically inactive version of <b>Cas9</b> and thus generate a <b>DNA binding protein</b>.</li>
     <li> ... construct a catalytically inactive version of <b>Cas9</b> and thus generate a <b>DNA binding protein</b>.</li>
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<li><i> ... In summary, we can now offer a universally applicable <b>toolkit</b> for gene regulation.</i></li>
<li><i> ... In summary, we can now offer a universally applicable <b>toolkit</b> for gene regulation.</i></li>
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6 opportunities with our uniCAS toolkit  
6 opportunities with our uniCAS toolkit  
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<p>
<p>
  We provide 3 different effectors, 2 methods & 1 effector controller! Using our toolkit it's possible to efficiently activate or repress genes. We also provide devices for effector controling by light. Use our custom-tailored <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/toolkit"> Manual Tool </a> to generate your individual manual for your needs of gene regulation. Further it's possible to target not only one, but multiple genes of interest! And we established <a id="link" href="">uniBAss</a> - our universal Binding Assay. Best of all: It's open source and in iGEM standard!  
  We provide 3 different effectors, 2 methods & 1 effector controller! Using our toolkit it's possible to efficiently activate or repress genes. We also provide devices for effector controling by light. Use our custom-tailored <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/toolkit"> Manual Tool </a> to generate your individual manual for your needs of gene regulation. Further it's possible to target not only one, but multiple genes of interest! And we established <a id="link" href="">uniBAss</a> - our universal Binding Assay. Best of all: It's open source and in iGEM standard!  
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dCas9 - The Heart of our toolkit  
dCas9 - The Heart of our toolkit  
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<!--simple DNA binding protein is the foundation of our project and all effectors used in this toolkit are fused to it.-->
<!--simple DNA binding protein is the foundation of our project and all effectors used in this toolkit are fused to it.-->
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Activation
Activation
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For activation we choosed VP-16 as effector. Through the transactivating function of VP-16 the expression of these genes will be enhanced.<br>
For activation we choosed VP-16 as effector. Through the transactivating function of VP-16 the expression of these genes will be enhanced.<br>
We achieved up to 30-fold activation.
We achieved up to 30-fold activation.
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Repression
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The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression. With this construct a strong repression could be observed.
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<!-- <img src="https://static.igem.org/mediawiki/2013/2/20/Freiburg2013-Highlights-KRAB3.png" style="width:500px"> -->
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Repression
 
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The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression. With this construct a strong repression could be observed.
 
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Specific chromatin modification was achieved by fusing the histone methyltransferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression.
Specific chromatin modification was achieved by fusing the histone methyltransferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression.
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Multiple Targeting
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One of the biggest advantages of the CRISPR/Cas9 system is that only one protein is required for targeting several DNA sites: For a new target there has to be just another guiding RNA. We designed an RNA plasmid, "<a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting">RNAimer</a>", containing this RNA. For multiple targeting different RNAimers can be easily combined using the iGEM BioBrick system.<br><br>
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And as the results show, multiple targeting is possible and even better!
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Multiple Targeting
 
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One of the biggest advantages of the CRISPR/Cas9 system is that only one protein is required for targeting several DNA sites: For a new target there has to be just another guiding RNA. We designed an RNA plasmid, "<a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting">RNAimer</a>", containing this RNA. For multiple targeting different RNAimers can be easily combined using the iGEM BioBrick system.<br><br>
 
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uniBAss
uniBAss
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We developed a novel and innovative ELISA based method to quantify the binding efficiency of our proteins. We called this binding assay uniBAss. This is a powerful tool for characterizing the modified dCas9 by assessing its DNA binding capacity with high throughput capabilities.
We developed a novel and innovative ELISA based method to quantify the binding efficiency of our proteins. We called this binding assay uniBAss. This is a powerful tool for characterizing the modified dCas9 by assessing its DNA binding capacity with high throughput capabilities.
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<td style="height:30px;"> <b>Figure 5: uniBAss - universal Binding Assay</b><br>
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<td style="height:30px; "> <b>Figure 5: uniBAss - universal Binding Assay</b><br>
First step ...  
First step ...  
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Latest revision as of 16:50, 4 October 2013


HIGHLIGHTS

  • In the last months we were able to ...

    • ... construct a catalytically inactive version of Cas9 and thus generate a DNA binding protein.
    • ... combine this modified dCas9 with different transcriptional effectors.
    • ... express this fusion proteins in various mammalian cell lines.
    • ... control mammalian gene expression via our modified CRISPR/Cas fusion proteins.
    • ... build devices for controling gene expression by light stimulus.
    • ... provide an RNA plasmid for easily inserting sequences for crRNAs which target every desired target.
    • ... build an online tool that generates customized manuals for using our toolkit
    • ... develop a method to assess the DNA binding capacity of our dCas9-fusion proteins.
    • ... make our dCas9 accessible to the whole iGEM community by mutating illegal iGEM restriction sites.
    • ... In summary, we can now offer a universally applicable toolkit for gene regulation.

  • 6 opportunities with our uniCAS toolkit

    We provide 3 different effectors, 2 methods & 1 effector controller! Using our toolkit it's possible to efficiently activate or repress genes. We also provide devices for effector controling by light. Use our custom-tailored Manual Tool to generate your individual manual for your needs of gene regulation. Further it's possible to target not only one, but multiple genes of interest! And we established uniBAss - our universal Binding Assay. Best of all: It's open source and in iGEM standard!

  • dCas9 - The Heart of our toolkit

    We started by mutating the DNA cleavage site in the Cas9 protein and generated a sequence specific DNA binding protein that is relying on a protein-RNA-DNA interaction. We are now able to influence the DNA binding locus and can direct the protein to requested DNA targets.
    This is the heart of our toolkit. A protein that allows multiple and sequence specifid DNA binding.

    For simple and flexible regulation we do need now effectors, that can be fused to the protein.

  • Activation

    For activation we choosed VP-16 as effector. Through the transactivating function of VP-16 the expression of these genes will be enhanced.
    We achieved up to 30-fold activation.

    Figure 1: Different error values plotted in increasing order.

  • Repression

    The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression. With this construct a strong repression could be observed.

    Figure 2: Different error values plotted in increasing order.

  • Chromatin modification (Repression)

    Specific chromatin modification was achieved by fusing the histone methyltransferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression.

    Figure 3: Different error values plotted in increasing order.

  • Multiple Targeting

    One of the biggest advantages of the CRISPR/Cas9 system is that only one protein is required for targeting several DNA sites: For a new target there has to be just another guiding RNA. We designed an RNA plasmid, "RNAimer", containing this RNA. For multiple targeting different RNAimers can be easily combined using the iGEM BioBrick system.

    And as the results show, multiple targeting is possible and even better!

    Figure 4: Different error values plotted in increasing order.

  • uniBAss

    We developed a novel and innovative ELISA based method to quantify the binding efficiency of our proteins. We called this binding assay uniBAss. This is a powerful tool for characterizing the modified dCas9 by assessing its DNA binding capacity with high throughput capabilities.

    Figure 5: uniBAss - universal Binding Assay
    First step ...

Retrieved from "http://2013.igem.org/Team:Freiburg/Highlights/2"