Team:Freiburg/Project/effector

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Activation

Transcriptional Activation via uniCAS-VP16

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.

Repression

Transcriptional Repression via uniCAS-KRAB

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 Fluorescence Microscopy and Flow Cytometry. Endogenuous 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.

Sources
(1) Bałan, B. and Słotwiński, R. (2008). VEGF and tumor angiogenesis. Centr Eur J Immunol 33, 232-236
(2) Birtle, Z. and Ponting, C. (2006). Meisetz and the birth of the KRAB motif. Bioinformatics 22, 2841-2845.
(3) Fritz, R. and Radziwill, G. (2011). CNK1 and other scaffolds for Akt/FoxO signaling. Biochimica et biophysica acta 1813, 1971-1977.
(4) Rosati, M. et al. (1991). Members of the zinc finger protein gene family sharing a conserved N-terminal module. Nucleic acids research 19, 5661-5667.
(5) Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome Biology 4, 4:231.
(6) Witzgall, R. et al. (1994). The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc Nati Acad Sci 91, 4514-4518.

Epigenetics

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 <p>
-Krüppel-associated Box (KRAB) repressor domains are highly conserved polypeptide motifs and were first functionally characterized in 1991 (<i>Rosati et al.</i>, 1991). Their occurence in about one third of all human zinc finger transcription factors (<i>Witzgall et al., 1994)</i> suggests key regulatory features in higher eukaryotic transcriptomics. In terms of tetrapod evolution, the role of their great abundance has been extensively discussed (<i>Birtle and Ponting</i>, 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 (<i>Urrutia</i>, 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.
+Krüppel-associated Box (KRAB) repressor domains are highly conserved polypeptide motifs and were first functionally characterized in 1991 (<i>Rosati et al.</i>, 1991). Their occurence in about one third of all human zinc finger transcription factors (<i>Witzgall et al.</i>, 1994) suggests key regulatory features in higher eukaryotic transcriptomics. In terms of tetrapod evolution, the role of their great abundance has been extensively discussed (<i>Birtle and Ponting</i>, 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 (<i>Urrutia</i>, 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.
-</p> <br>
+</p><br>
 <p>
-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 (<i>Fritz and Radziwill</i>, 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 Fluorescence Microscopy and Flow Cytometry. Endogenuous levels of VEGF-A, a key factor in hypoxic tumor angiogenesis (<i>Bałan and Słotwiński, 2008</i>), were also successfully reduced and quantified through an Enzyme-Linked Immunosorbent Assay.
+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 (<i>Fritz and Radziwill</i>, 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 Fluorescence Microscopy and Flow Cytometry. Endogenuous levels of VEGF-A, a key factor in hypoxic tumor angiogenesis (<i>Bałan and Słotwiński</i>, 2008), were also successfully reduced and quantified through an Enzyme-Linked Immunosorbent Assay.
 </p> <br></div>