Folie 1

Student Members: M. Agne, I. Blank, A. Emhardt, C. Gäbelein, F. Gawlas, N. Gillich, P. Gonschorek, T. Juretschke, S. Krämer, N. Louis, A. Müller, A. Rudorf, L. Schäfer, M. Scheidmann, L. Schmunk, P. Schwenk, M. Stammnitz, P. Warmer
Contact: freiburg.igem2013@gmail.com
Supervisors: A. Fischer, B. Kaufmann, H. Wagner, J. Kaiser, G. Radziwill, W. Weber
Our team developed a universal toolkit, termed uniCAS, that enables customizable
gene regulation in mammalian cells. Therefore, we engineered the recently
understood and highly promising Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR) system. The CRISPR-associated catalytically inactive
dCas9 is the heart of our toolkit: A protein that enables multiple and sequence -
We fused dCas9 to the transactivation domain of VP16.
This fusion protein was successfully used to activate
gene expression of the secreted embryonic alkaline
phosphatase (SEAP) reporter in mammalian cells in
presence of appropriate crRNAs. We achieved over 25fold upregulation of SEAP expression (see Figure 1 C).
A
specific DNA binding. The key components for binding dCas9 to its targets are two
small, non-coding RNAs: The CRISPR-RNA (crRNA) and the transactivating crRNA
(tracrRNA). The RNA complex guides dCas9 to the DNA sequences of interest. By
fusing several effector domains to dCas9, we constructed novel engineered
proteins for gene regulation, even controllable by light stimulus.
Specific chromatin modification was achieved by fusing the
histone methyltransferase G9a to dCas9 and thereby resulting
in an epigenetic BioBrick. G9a primarily methylates histone 3.
Different endogenous loci in the vascular endothelial growth
factor (VEGF) gene were targeted in mammalian cells. This
resulted in an up to 50% repression (*, p<0.05) (see Figure 2 C).
A
C
C
Target sites
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The greatest advantage of the CRISPR/Cas9
system is that only one protein is required for
targeting various DNA sequences. The only
component which needs to be replaced is the
CRISPR-RNA (crRNA). We therefore designed
an RNA plasmid termed the RNAimer. It
provides the backbone for easy insertion of
the crRNA sequences.
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B
Figure 7: Plasmid design of the RNAimer. Transactivating crRNA is
transcribed by H1 promoter and crRNA by U6 promoter. The crRNA can
be exchanged by cutting the plasmid with Bbs1.
Me
B
VEGF
SEAP
Figure 1. Activation of SEAP in mammalian cells using the fusion protein dCas9-VP16. (A)
Plasmid design for activating gene expression. (B) Schematic representation of dCas9-VP16
activation system. (C) Plasmids were cotransfected into HEK-293T cells and expression of
SEAP reporter was measured after 24 h. The numbers characterizing the crRNAs represent
the distance from the translation start site.
Figure 2. Repression of the endogenous VEGF gene via histone modification by dCas9-G9a. (A)
Plasmid design for histone modification. (B) Schematic representation of dCas9-G9a repression
system. (C) Plasmids shown in A were cotransfected into HEK-293T cells and the amount of VEGF
expression was measured. VEGF-8 corresponds to a region in the VEGF locus at position -8 bp from
the transcription start site (TSS) and VEGF-573 corresponds to a region -573 bp from the TSS.
The transcriptional repressor domain Krüppel-associated
box (KRAB) was fused to dCas9 resulting in a fusion
protein with the ability to repress gene expression. The
device was tested in mammalian cells by targeting
different sites in the endogenous VEGF locus. Up to 50%
repression was achieved (Figure 3).
A
For multiple targeting, different crRNAs can
be combined into one RNAimer plasmid
using the BioBrick standard assembly
method. Gene regulation in mammalian cells
worked even more efficiently when using
multiple targets (see Figure 1 C).
We aimed to control our system with light
thereby allowing for gene regulation with
high spatiotemporal resolution. We
engineered a system for induction by red,
UVB and blue light. The blue light system is
based on the light-triggered interaction of
CRY2 and CIB1. CRY2 was fused to dCas9
which can upon light-stimulus recruit the
CIB1-VP16 fusion protein to any DNA
sequence of interest.
D
B
VEGF
C
GFP
A
GFP
dCas9-KRAB
Target sites
Figure 3: Repression of GFP & the endogenous VEGF gene expression via dCas9-KRAB.
(A) Plasmid design for repression. (B) Schematic representation of the dCas9-KRAB
repression system. (C) Repression of GFP in HeLa cells via dCas9-KRAB analyzed by
fluorescence microscopy. (D) HEK-293T cells were cotransfected with plasmids shown in A
and the amount of VEGF expression was measured. The numbers characterizing the
crRNAs represent the distance from the transcription start site.
We used a thermodynamic approach
to model our system for dCas9-VP16
gene activation.
Figure 4: Behavior of the different components. By minimizing the least square error
between the time resolved dCas9 and SEAP data (purple) and the model prediction (cyan)
the best parameters for our model were found. Using these parameters a potential
behavior for measured and unmeasured (tracr/crRNA & gene recognition complex)
components can be predicted. Expression of our fusion proteins seems to represent the
bottleneck part of the system.
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B
We developed a novel and innovative ELISA-based method to
quantify the binding efficiencies of our dCas9 fusion proteins:
The uniCAS Binding Assay (uniBAss). For this purpose,
biotinylated oligos were coated on 96-well ELISA plates via the
interaction with streptavidin.
A
B
SEAP
C
Figure 5. The uniCAS Binding Assay (uniBAss). (A) Schematic overview of uniBAss. (B) Binding affinity
of dCas9 fusion constructs. HEK-293T cells were cotransfected with the indicated dCas9 constructs
and a crRNA plasmid. Samples were normalized to dCas9 expression levels that were quantified by
Western Blot. Background level of wells without coated oligos was substracted of depicted values.
Gene regulation was achieved based on a modified CRISPR/Cas9 system. We
engineered a toolkit which offers efficient and customizable gene activation and
repression via effector domains fused to the dCas9 protein. Combining uniCAS
with multiple targeting, light induction and the uniBAss we generated a universally
1
Figure 6. Light controlled
activation of SEAP reporter via
dCas9-CRY2 and CIB1-VP16. (A)
Plasmid design for light control.
(B) Schematic representation of
the dCas9-CRY2 & CIB1-VP16 light
system. (C) Transfection of
plasmids (Figure 6 A) in NIH/3T3
cells and illumination with 460
nm. A 5-fold upregulation of SEAP
reporter was achieved (*, p<0.05).
dCas9-UVR8 was targeted to four
DNA regions upstream of the
promoter.
applicable toolkit which allows regulation of any gene of interest.
Our toolbox of standardized parts of the CRISPR/Cas9 system
offers broad application in research fields such as tissue
engineering, stem cell reprogramming and fundamental research.