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Team:Freiburg/Highlights
From 2013.igem.org
HIGHLIGHTS
In the last months we were able to...
- ...constuct a catalytically inactive version of Cas9 and this way generate a new class of DNA binding proteins.
- ...combine this modified dCas9 with different transcriptional effectors.
- ...express this system in various mammalian cell lines.
- ...control human gene expression via our modified CRISPR/Cas system.
- ...regulate gene expression on light stimulus.
- ...make our dCas9 accesible to the whole iGEM comunity by mutating illegal iGEM restriction sites.
In summary we build up a universal toolkit for gene regulation.
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6 opportunities to customize your experiments We provide 3 different effectors, 2 methods and 1 effector controler that allows either to effectively repress or activate genes - also on stimulus. Use our custom-tailored Manual Tool to generate individual manuals. Best of all: Its all open source and in iGEM standard! |
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dCas9 - Heart of our toolkit
We started by mutating the DNA cleavage site in the Cas9 protein and generated a DNA binding protein that is relying on a RNA-DNA interaction. This 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 The activation domain VP16 is able to activate transcription of genes. |
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Repression The fusion of the transcriptional repressor domain KRAB leads to synthetic repression of gene expression. |
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Chromatin modification (Repression) Specific chromatin modification was achieved by fusing a histone methyltransferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression. |
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uniBAss - Binding Assay We developed an ELISA based method. With this method we can 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. |
