"
Team:Freiburg/Highlights
From 2013.igem.org
- ...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.
We started with mutating the nickase sites in the Cas9 protein and could generate a novel type of DNA binding protein that is relying on protein-RNA-DNA interaction.
By fusing effector domains to Cas9 gives rise to new properties. The activation domain VP16 was able to activate transcription of genes. The fusion of the transcriptional repressor domain KRAB led to decreasing gene expression. Specific chromatin modification was achieved by fusing a histone methyl transferase G9a to dCas9. With this protein we are able to specifically repress endogenous gene expression.
Utilizing the light receptor system of higher plants we were able to dimerize the dCAS9 with the effector domain on light stimulus and in consequence activate the system.
By building a plasmid containing the necessary RNAs and insertion sites for targeting we achieved 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 approach, that is able to give a quantitative impression of binding strength of dCAS9 to a given target. 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.
To sum it all uo able to establish a new modularized tool kit for modulating gene expression: The uniCAS Toolkit!
