Team:Freiburg/Highlights

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HIGHLIGHTS
 
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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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-
    <li> ... construct a catalytically inactive version of <b>Cas9</b> and thus generate a <b>DNA binding protein</b>.</li>
 
-
    <li> ... combine this modified dCas9 with different transcriptional <b>effectors</b>.</li>
 
-
    <li> ... express this fusion proteins in various <b>mammalian</b> cell lines.</li>
 
-
    <li> ... <b>control</b> mammalian gene expression via our modified CRISPR/Cas fusion proteins.</li>
 
-
    <li> ... build devices for controling gene expression by <b>light stimulus</b>.</li>
 
-
<li> ... provide an RNA plasmid for easily inserting sequences for crRNAs which target every desired target DNA.</li>
 
-
<li> ... target multiple DNA sequences with only inserting this one RNA plasmid.</li>
 
-
<li> ... build an online tool that generates customized <b>manuals</b> for using our toolkit</li>
 
-
    <li> ... develop a method to assess the <b>DNA binding capacity</b> of our dCas9-fusion proteins.</li>
 
-
    <li>... make our dCas9 <b>accessible</b> to the whole iGEM community by mutating illegal iGEM restriction sites</b>.</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
 
-
</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!
 
-
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-
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-
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CAS 9
 
-
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HIGHLIGHTS
 +
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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> ... combine this modified dCas9 with different transcriptional <b>effectors</b>.</li>
 +
    <li> ... express this fusion proteins in various <b>mammalian</b> cell lines.</li>
 +
    <li> ... <b>control</b> mammalian gene expression via our modified CRISPR/Cas fusion proteins.</li>
 +
    <li> ... build devices for controling gene expression by <b>light stimulus</b>.</li>
 +
<li> ... provide an RNA plasmid for easily inserting sequences for crRNAs which target every desired target.</li>
 +
<li> ... build an online tool that generates customized <b>manuals</b> for using our toolkit</li>
 +
    <li> ... develop a method to assess the <b>DNA binding capacity</b> of our dCas9-fusion proteins.</li>
 +
    <li>... make our dCas9 <b>accessible</b> to the whole iGEM community by mutating illegal iGEM restriction sites</b>.</li>
 +
<li><i> ... In summary, we can now offer a universally applicable <b>toolkit</b> for gene regulation.</i></li>
 +
</ul>
 +
</div>                     
 +
                        <div class=clear></div>
 +
                    </div>
 +
                </li>
 +
             
 +
<li>
 +
                    <div class="frontpage-slide">  <div id="left_column">  
-
</div>
 
-
</td>
 
-
<td>
 
-
<div id="right_column">
 
<p id="headline">  
<p id="headline">  
 +
6 opportunities with our uniCAS toolkit
 +
</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!
 +
</p> 
 +
</div>
 +
                        <div class="slide-right-col"><div id="right_column">               
 +
<img id="main_images" src="https://static.igem.org/mediawiki/2013/f/f5/Freiburg2013-toolkit-for-highlight2.png" style="width:280px; margin-left:100px; margin-top:-10px;">
 +
                         
 +
                        </div></div>
 +
                        <div class=clear></div>
 +
                    </div>
 +
                </li>
 +
                <li>
 +
                    <div class="frontpage-slide">
 +
                        <div id="left_column"> <p id="headline">
dCas9 - The Heart of our toolkit  
dCas9 - The Heart of our toolkit  
</p>
</p>
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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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<img src="https://static.igem.org/mediawiki/2013/f/f4/Dna_freiburg_13_ruppel_die_katz.png" style="width:650px; margin-top:110px; margin-left:-106px;">
 
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 +
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 +
                        <div class="slide-right-col" style="margin-top:-450px">
 +
                            <div id="right_column"> <div style="text-align: center;">
 +
<!-- Cas video -->
 +
</div>
-
 
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-
<!-- **************************************************************************************************************************************************
+
                        <div class=clear></div>
-
 
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-
ACTIVATION
+
                </li>    
-
 
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-
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<p id="headline">  
<p id="headline">  
Activation
Activation
</p>
</p>
-
<p style="padding-right:30px;">
+
<p>
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.
-
</p>
+
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+
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 +
Repression
 +
</p>
 +
<p>
 +
The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression. With this construct a strong repression could be observed.
 +
</p>
-
<!-- **************************************************************************************************************************************************
+
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-
 
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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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EPIGENETICS
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Chromatin modification (Repression)
Chromatin modification (Repression)
</p>
</p>
-
<p style="padding-right:30px;">
+
<p >
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.
</p>
</p>
-
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<p id="headline" >
Multiple Targeting
Multiple Targeting
 +
</p>
 +
<p>
 +
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>
 +
And as the results show, multiple targeting is possible and even better!
 +
</p>
-
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-
Multiple Targeting
 
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</p>
 
-
<p style="padding-left:60px;">
 
-
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>
 
-
And as the results show, multiple targeting is possible and even better!
 
-
</p>
 
</div>
</div>
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 +
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 +
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UNIBASS
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uniBAss
uniBAss
</p>
</p>
-
<p style="padding-right:30px;">
+
<p>
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.
</p>
</p>
 +
             
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<EMBED  HEIGHT=362 WIDTH=400 TYPE="video/quicktime" 
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<img style="width:400px; margin-left:-20px; margin-top:10px;" src="https://static.igem.org/mediawiki/2013/f/f6/UniBASS_Freiburg_2013.JPG" >
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PLUGINSPAGE="http://www.apple.com/quicktime/download/"  
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-
<td style="height:30px; margin-left:-150px; "> <b>Figure 5: uniBAss - universal Binding Assay</b><br>
+
<td style="height:30px; "> <b>Figure 5: uniBAss - universal Binding Assay</b><br>
First step ...  
First step ...  
</td>
</td>
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6 Reasons for loving the uniCAS toolkit
+
-
</p>
+
-
<p>
+
-
All you need to realize your personal experiments are 6 smart amazingly easy to use tools that come with the uniCAS toolkit. Just use our custom-tailored <a id="link" href="https://2013.igem.org/Team:Freiburg/Project/toolkit"> uniTOOL </a> to generate individual manuals. Best of all: It´s all open source and non commercial!
+
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<p id="h1">Highlights
 
-
</p>
 
-
 
-
<p id="h2">In the last months we were able to...</p>
 
-
 
-
<ul style="font-size:20px">
 
-
 
-
    <li>...constuct a <b>catalytically inactive version of Cas9</b> and this way generate a new class of DNA binding proteins.</li>
 
-
    <li>...combine this modified dCas9 with <b>different transcriptional effectors</b>.</li>
 
-
    <li>...express this system in various <b>mammalian cell lines</b>.</li>
 
-
    <li>...control human <b>gene expression</b> via our modified CRISPR/Cas system.</li>
 
-
    <li>...regulate gene expression on <b>light stimulus</b>.</li>
 
-
    <li>...make our <b>dCas9 accesible to the whole iGEM comunity</b> by mutating illegal iGEM restriction sites</b>.</li>
 
-
<br>
 
-
In summary we build up a <b>universal toolkit for gene regulation</b>.
 
-
 
-
 
-
</ul>
 
-
 
-
<p id="h3">In the beginning
 
-
</p>
 
-
<p>
 
-
A mutated Cas9 derived protein without nickase function was our start. This is basically a DNA binding protein, that is relying on a <b>protein-RNA-DNA</b>
 
-
interaction.
 
-
</p><p></p><p>
 
-
By fusing <b>effector domains</b> to dCas9 we altered the properties in various ways.</p>
 
-
 
-
<p id="h3">Activation
 
-
</p>
 
-
 
-
<div style="position:absolute; top:1000px; left:550px;"> <img style="height:200px" src="https://static.igem.org/mediawiki/2013/f/f4/Freiburg_2013_highlights_Activation-Pfeil.png"> </div>
 
-
<p> The <b>activation domain VP16</b> is able to activate transcription of
 
-
 
-
genes.<p></p>
 
-
<div>
 
-
<table class="imgtxt" width="500px">
 
-
<tr>
 
-
<td> <img class="imgtxt" width="500px" src="https://static.igem.org/mediawiki/2013/5/59/VP16_Highlights_Freiburg_2013.JPG"> </td>
 
-
</tr>
 
-
<tr>
 
-
<td> <b>Figure 1: Activation by Cas9:VP16 </b><br>
 
-
By fusing the transcriptional activation domain VP16 to dCas9, we are able to activate a SEAP reporter transcription.
 
-
</td>
 
-
</tr>
 
-
</table>
 
-
</div>
 
-
 
-
<p id="h3"> Repression
 
-
</p>
 
-
 
-
<div style="position:absolute; top:1550px; left:550px;"> <img style="height:200px" src="https://static.igem.org/mediawiki/2013/2/2f/Freiburg_2013_highlights_Repression-Pfeil.png"> </div>
 
-
 
-
 
-
The fusion of the <b>transcriptional repressor domain KRAB</b> leads to synthetic repression of gene expression.<p></p>
 
-
 
-
<div>
 
-
<table class="imgtxt" width="500px">
 
-
<tr>
 
-
<td> <img class="imgtxt" width="500px" src="https://static.igem.org/mediawiki/2013/5/52/KRAB_Highlights_Freiburg_2013.JPG"> </td>
 
-
</tr>
 
-
<tr>
 
-
<td> <b>Figure 2: Repression via dCas9:KRAB </b><br>
 
-
Using dCas9:KRAB we were able to repress GFP expression in mammalian cells.
 
-
</td>
 
-
</tr>
 
-
</table>
 
-
</div>
 
-
 
-
<p id="h3">Chromatin modification (Repression)
 
-
</p>
 
-
 
-
<div style="position:absolute; top:2050px; left:550px;"> <img style="height:200px" src="https://static.igem.org/mediawiki/2013/2/2f/Freiburg_2013_highlights_Repression-Pfeil.png"> </div>
 
-
 
-
 
-
Specific <b>chromatin modification</b> was
 
-
 
-
achieved by fusing a histone methyltransferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression. </p> <p>
 
-
 
-
 
-
<div>
 
-
<table class="imgtxt" width="500px">
 
-
<tr>
 
-
<td> <img class="imgtxt" width="500px" src="https://static.igem.org/mediawiki/2013/0/0d/G9a_Highlights_Freiburg_2013.JPG"> </td>
 
-
</tr>
 
-
<tr>
 
-
<td> <b>Figure 3: Endogenous, stable repression by dCas9:G9a </b><br>
 
-
Chromatin remodeling, resulting in repression of endogenous genes is possible by fusing the histone methyltransferase G9a to dCas9.
 
-
</td>
 
-
</tr>
 
-
</table>
 
-
</div>
 
-
 
-
</p> <p> </p> <p>
 
-
 
-
<p id="h3">Light switch
 
-
</p>
 
-
 
-
We were able to induce our system on <b>light stimulus</b>. This was possible by using photoreceptors of higher plants.
 
-
</p> <p> </p> <p>
 
-
 
-
<p id="h3">Targeting with RNAimer
 
-
</p>
 
-
By building a plasmid containing the necessary<b> RNAs</b> and <b>insertion sites</b> for targeting we created a modular, BioBrick compatible system for <b>multiple
 
-
 
-
DNA targeting: The RNAimer.</b>
 
-
Using our RNAimer plasmid it is easy to combine several target sequences on one plasmid using the BioBrick standard.
 
-
</p> <p> </p> <p>
 
-
 
-
<p id="h3">uniBAss - Binding Assay
 
-
</p>
 
-
We developed an ELISA based method. With this method we can quantify the <b>binding efficiency </b>of our proteins. We called this binding assay <b>uniBAss</b>. It is
 
-
 
-
a powerful tool for characterizing the modified dCas9 by assessing its DNA binding capacity.
 
-
</p> <p>
 
-
<div>
 
-
<table class="imgtxt" width="500px">
 
-
<tr>
 
-
<td> <img class="imgtxt" width="500px" src="https://static.igem.org/mediawiki/2013/f/f6/UniBASS_Freiburg_2013.JPG"> </td>
 
-
</tr>
 
-
<tr>
 
-
<td> <b>Figure 4: uniBAss </b><br>
 
-
We developed an assay for testing the binding capacity of our constructs.
 
-
</td>
 
-
</tr>
 
-
</table>
 
-
</div>
 
-
</p> <p>
 
-
<p id="h3">Conclusion
 
-
</p>
 
-
<p>We established a new modularized toolkit for modulating gene expression specifically: <b>The uniCAS Toolkit!</b></p><br>
 
-
<img src="https://static.igem.org/mediawiki/2013/d/de/Toolkit_Projekt_Freiburg_2013.PNG" width="700px">
 
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Revision as of 15:00, 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 ...

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