Team:Freiburg/Project/effector

Transcriptional Activation via uniCAS-VP16 System in Mammalian Cells

The ability to specifically control transcription is a valuable tool to study gene function, to construct synthetic gene networks with desired properties and even to combat diseases. Zinc-finger proteins (ZFPs) and transcription activator-like effectors (TALEs) comprise a powerful class of tools for genomic engineering so far. However, CRISPR/Cas (clustered regulary interspaced short palindromic repeats/ CRISPR associated) is a recently discovered adaptive immune system that protects bacteria and archaea against invading DNA (viruses and plasmids) by cleaving foreign nucleic acids in a sequence-specific manner. Recent studies revealed the potential of the type II CRISPR/Cas system that can be engineered to target desired DNA sequences. It is more scalable, affordable and easier to engineer compared to ZFPs and TALEs. The aim of this subproject was to engineer a new form of activation system based on a CRISPR RNA (crRNA)-guided Cas9-VP16 fusion protein which is able to activate gene expression upon a gene reporter construct. Therefore the CRISPR/Cas system II was modified whereby the VP16 trans-activation domain of the herpes simplex virus was fused to a catalytically inactive Cas9. For targeting a variety of different loci, various crRNAs were designed to guide the fusion protein Cas9-VP16 to its cognate target. The fusion protein is guided to desired DNA sequences by a co-expressed crRNA. Linking of functional modified Cas9 to a transcriptional activator domain can effectively upregulate gene expression of gene reporter constructs. Cells co-expressing the Cas9-VP16 fusion protein and reporter construct, exhibit an increase in SEAP production up to 10-fold. CONTENT CONTENT