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Team:Freiburg/Highlights
From 2013.igem.org
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<div id="h1">Highlights | <div id="h1">Highlights | ||
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<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. | + | 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> | </p><p></p><p> | ||
| - | By fusing <b>effector domains</b> to Cas9 we altered the properties it in various ways.</p><p> The <b>activation domain VP16</b> is able to activate transcription of genes. The fusion of the <b>transcriptional repressor domain KRAB</b> leads to synthetic repressor of gene expression. Specific <b>chromatin modification</b> was achieved by fusing a histone methyl transferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression. </p> <p> </p> <p> | + | By fusing <b>effector domains</b> to Cas9 we altered the properties it in various ways.</p><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=""> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <b>Figure 1: Bildunterüberschrift </b><br> | ||
| + | HIER DANN ALLES ANDERE | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | |||
| + | The fusion of the <b>transcriptional repressor domain KRAB</b> leads to synthetic repressor of gene expression.<p></p> | ||
| + | |||
| + | <div> | ||
| + | <table class="imgtxt" width="500px"> | ||
| + | <tr> | ||
| + | <td> <img class="imgtxt" width="500px" src=""> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <b>Figure 1: Bildunterüberschrift </b><br> | ||
| + | HIER DANN ALLES ANDERE | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | |||
| + | Specific <b>chromatin modification</b> was | ||
| + | |||
| + | achieved by fusing a histone methyl transferase 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=""> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <b>Figure 1: Bildunterüberschrift </b><br> | ||
| + | HIER DANN ALLES ANDERE | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | |||
| + | </p> <p> </p> <p> | ||
We were able to induce our system on <b>light stimulus</b>. This was possible by using photorecetors of higher plants. | We were able to induce our system on <b>light stimulus</b>. This was possible by using photorecetors of higher plants. | ||
</p> <p> </p> <p> | </p> <p> </p> <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> | + | 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. | Using our RNAimer plasmid it is easy to combine several target sequences on one plasmid using the BioBrick standard. | ||
</p> <p> </p> <p> | </p> <p> </p> <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 the characterization of the interaction between the modified Cas9 and the locus specific RNA. | + | 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 |
| - | </p> <p> </p> <p> | + | |
| + | a powerful tool for the characterization of the interaction between the modified Cas9 and the locus specific RNA. | ||
| + | </p> <p> | ||
| + | <div> | ||
| + | <table class="imgtxt" width="500px"> | ||
| + | <tr> | ||
| + | <td> <img class="imgtxt" width="500px" src=""> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <b>Figure 1: Bildunterüberschrift </b><br> | ||
| + | HIER DANN ALLES ANDERE | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | </p> <p> | ||
| - | In summary, we established a new modularized tool kit for modulating gene expression: <b>The uniCAS Toolkit!</b | + | In summary, we established a new modularized tool kit for modulating gene expression: <b>The uniCAS Toolkit!</b> |
| - | + | ||
</div> | </div> | ||
Revision as of 15:32, 29 September 2013
Highlights
In the last months we were able...
- ...to design a catalytically inactive version of Cas9 and designing a new class of DNA binding proteins.
- ...to combine this modified Cas9 with different effectors.
- ...to express the system in various mammalian cell lines.
- ...to control human gene expression via our modified CRISPR/Cas system.
- ...to control gene expression on light stimulus.
A mutated Cas9 derived protein without nickase function was our start. This is basically a DNA binding protein, that is relying on a protein-RNA-DNA interaction.
By fusing effector domains to Cas9 we altered the properties it in various ways.
The activation domain VP16 is able to activate transcription of genes.
| |
| Figure 1: Bildunterüberschrift HIER DANN ALLES ANDERE |
| |
| Figure 1: Bildunterüberschrift HIER DANN ALLES ANDERE |
| |
| Figure 1: Bildunterüberschrift HIER DANN ALLES ANDERE |
We were able to induce our system on light stimulus. This was possible by using photorecetors of higher plants.
By building a plasmid containing the necessary RNAs and insertion sites for targeting we created a modular, BioBrick compatible system for multiple DNA targeting: The RNAimer. Using our RNAimer plasmid it is easy to combine several target sequences on one plasmid using the BioBrick standard.
We developed an ELISA based method. With this method we can quantify the binding efficiency of our proteins. We called this binding assay uniBAss. It is a powerful tool for the characterization of the interaction between the modified Cas9 and the locus specific RNA.
| |
| Figure 1: Bildunterüberschrift HIER DANN ALLES ANDERE |
In summary, we established a new modularized tool kit for modulating gene expression: The uniCAS Toolkit!
