Team:Freiburg/Project/effector

Krüppel-associated Box (KRAB) repressor domains are highly conserved polypeptide motifs and were first functionally characterized in 1991 (Rosati et al., 1991). Their occurence in about one third of all human zinc finger transcription factors (Witzgall et al., 1994) suggests key regulatory features in higher eukaryotic transcriptomics. In terms of tetrapod evolution, the role of their great abundance has been extensively discussed (Birtle and Ponting, 2006). Even though KRAB minimal domains are usually no longer than ~ 50-75 amino acids, their mechanism of function remains complex. Common biochemical models suggest a key role in epigenetic silencing, by recruiting a scaffold of diverse proteins to the zinc fingers‘ binding site - amongst others histone deacetylases and histone methyltransferases (Urrutia, 2004). Til date, KRAB domains were attached to several DNA binding proteins such as lacR and tetR, thereby silencing gene expression downstream of designed reporter targets.

In this work, KRAB was fused to enzymatically inoperable dCas9. Thus, a transcriptional repressor with the flexibility to target almost any DNA sequence of interest was yielded. Transient SEAP expression could thus be reduced by almost 60 %. In a second attempt, CMV-driven expression of the signaling scaffold protein CNK1 (Fritz and Radziwill, 2011) was targeted over 36h - being partially knocked down to background amounts. Furthermore, GFP reporter expression was shown to be drastically reduced by dCas9-KRAB in both Fluorescence Microscopy and Flow Cytometry data. Endogenous levels of VEGF-A, a key factor in hypoxic tumor angiogenesis (Bałan and Słotwiński, 2008), were also successfully reduced and quantified through an Enzyme-Linked Immunosorbent Assay.