... construct a catalytically inactive version of Cas9 and thus generate a DNA binding protein.
... combine this modified dCas9 with different transcriptional effectors.
... express this system in various mammalian cell lines.
... control mammalian gene expression via our modified CRISPR/Cas system.
... regulate gene expression on light stimulus.
... 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.
... build an online tool that generates customized manuals for using our toolkit.
... 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 DNA binding protein that is relying on a Protein-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.
Activation
The activation domain VP16 is able to activate transcription of genes. We achieved up to 30-fold activation.
Figure 8: 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.
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.
uniBAss - Binding Assay
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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