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Team:Freiburg/Project/induction
From 2013.igem.org
Light
Introduction
Up to the present day, various networks to control cellular behavior have been developed. Small molecules such as antibiotics are often used as an inducer (Weber et al. 2002; Jakobus et al. 2012). However these systems often lack reversibility, exact spatial control and show toxic and pleiotropic side effects (Yang et al. 2013; Ortiz et al. 2013). These problems can be solved using for instance optogenetic tools to control gene expression (Müller et al. 2013a; Müller et al. 2013b) or other desired interactions (Levskaya et al.), e.g.activation of protein splicing (Tyszkiewicz and Muir 2008). The precise control of gene expression is of high value for basic research or further applications such as gene therapy.
The field of optogenetics made great progress within the past few years. From the discovery of new mechanisms like the PhyB-PIF interaction (Min Ni, et al. 1998) to the practical use in synthetic biology (Shimizu-Sato et al. 2002) only few years passed. The recently emerged CRISPR/Cas system established in mammalian cells allowed first applications in 2011 (Jinek et al. 2012). The aim of this study is to connect both fields to form a new system for the use in mammalian cells. The combination of Cas9 with optogenetic tools has potential use for in multiple applications enabling accurate genome editing.
Biotic and abiotic influences contribute major impact on evolution. All living beings are able to sense the state of their surrounding environment to a certain extent. Sensing light is crucial for most organisms. Sensing light is crucial for most organisms. For plants, light serves not only as orientation, but mainly as an energy source. Plants have developed mechanisms to perceive the intensity, periodicity and wavelength of light. The perception of light is directly linked to the metabolism. A unique set of photoreceptors is allowing them to sense exact quantities and qualities of light (Lariguet and Dunand 2005).
Our universal toolkit will allow you to use the full spektrum of light as induction signal. All proteins are derived from ''Arabidopsis thaliana''. Phytochrome B is responsible for red-light induction. Cryprochrome 2 is respinsible for blue-light induction and the UV-light receptor UVR8 is responsible for induction of UV-B light.
Induction by a red-light stimulus
One of the best described Phytochromes is Phytochrome B (PhyB). In Arabidopsis thaliana, PhyB predominantly controls seedling establishment. PhyB is a protein with a molecular mass of 125kDa that is predominantly located in the cytoplasma. Illumination with red light (660nm wavelength) leads to binding of Phytochrome Interacion Factor 6 (PIF6). The interaction can be abolished by illumination with far-red light (740nm) wavelength.A framework using the unique properties of PhyB and PIF6 was designed to enable the recruitment of different effector proteins to any desired gene locus. The system was designed to work as described: Fusion proteins linking Cas9 to PIF6 were designed, as well as fusion proteins linking the interaction partner of PIF6 – PhyB – to several effector molecules such as VP16 or KRAB.
References
Jakobus, Kathrin; Wend, Sabrina; Weber, Wilfried (Chemical Society Reviews; 2012): Synthetic mammalian gene networks as a blueprint for the design of interactive biohybrid materials.Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. (Science; 2012): A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.
Lariguet, Patricia; Dunand, Christophe (Journal of Molecular Evolution; 2005): Plant Photoreceptors: Phylogenetic Overview (Journal of Molecular Evolution).
Min Ni, James M. Tepperman, and Peter H. Quail* (Cell; 1998): PIF3, a Phytochrome-Interacting Factor Necessary for Normal Photoinduced Signal Transduction, Is a Novel Basic Helix-Loop-Helix Protein.
Muller, K.; Engesser, R.; Metzger, S.; Schulz, S.; Kampf, M. M.; Busacker, M. et al. (Nucleid Acids Research; 2013a): A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells.
Muller, K.; Engesser, R.; Schulz, S.; Steinberg, T.; Tomakidi, P.; Weber, C. C. et al. (Nucleid Acids Research; 2013b): Multi-chromatic control of mammalian gene expression and signaling.
Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf (Genesis; 2013): Reversible and tissue-specific activation of MAP kinase signaling by tamoxifen in braf V637 ER T2 mice.
Shimizu-Sato, Sae; Huq, Enamul; Tepperman, James M.; Quail, Peter H. (Nature Biotech, 2002): A light-switchable gene promoter system.
Tyszkiewicz, Amy B.; Muir, Tom W. (Nature Methods; 2008): Activation of protein splicing with light in yeast.
Weber, Wilfried; Fux, Cornelia; Daoud-El Baba, Marie; Keller, Bettina; Weber, Cornelia C.; Kramer, Beat P. et al. (Nature Biotechnology; 2002): Macrolide-based transgene control in mammalian cells and mice.
Yang, Geniey; Nowsheen, Somaira; Aziz, Khaled; Georgakilas, Alexandros G. (Pharmacology & Therapeutics; 2013): Toxicity and adverse effects of Tamoxifen and other anti-estrogen drugs.
Hormon
Introduction
The aim of this project was to control Cas9 (CRISPR associated protein 9) activity by an external 4-OHT (4-hydroxytamoxifen) stimulus: Only when 4- OHT is applied, Cas9 should enter the nucleus. Thus, Cas9 was fused to the 4-OHT inducible receptor domain ERT2 (mutated hormone binding domain of the human estrogenic receptor). To facilitate the detection of the fusion protein a HA tag and the fluorescent protein mCherry were added.
The native estrogenic receptor (ER) acts as an enhancer: Upon hormone binding this protein stimulates the transcription of specific genes. It consists
mainly of a DNA and a hormone binding domain (HBD) (KUMAR ET AL. 1987).
Steroid receptors as ER require chaperones (mostly Hsp90) to be functional: They open the hydrophobic hormone binding cleft and carry them after
binding of the appropriate ligand with the help of immunophilins along the cytosceleton to the nucleus (PRATT & TOFT 2003). For the glucocorticoid
receptor (GR) this interaction is crucial for nuclear import (CZAR ET AL. 1997). Even within the nucleus most steroid receptors are in complex with Hsp90
(SMITH & TOFT 2008). Without a ligand steroid receptors move continuously into and out of the nucleus. When the adequate hormone is bound GR
localization becomes nuclear within a half life of about 4,5 min (PRATT & TOFT 2003).
Tamoxifen is a competitive inhibitor of ER. For that reason it is widely used for treatment of breast cancer (FURR & JORDAN 1984). In the body of higher
vertebrates Tamoxifen becomes metabolized to 4-OHT (BORGNA ET AL. 1980). This form is more potent in inhibiting the ER which leads to a stronger
repression of estrogen-dependent cell growth (CARDOSO ET AL. 2003).
In 1993 DANIELIAN ET AL. mutated the murine ER, so that it is no more sensitive to estrogen, but becomes active after binding of tamoxifen. LITTLEWOOD
ET AL. (1995) took the HBD of this mutated ER and fused it to the c-Myc protein. This way they were able to control Myc-induced proliferation and
apoptosis. Later on the DNA recombinase Cre was fused with a more tamoxifen sensitive human HBD mutant named ERT2 (FEIL ET AL. 1997). For the
regulation 4-OHT is preferred to tamoxifen because it yields a higher effect with lower concentrations (ZHANG ET AL. 1996).
Besides the inhibition of the ER, tamoxifen can decrease the cellular ATP level via uncoupling of the mitochondrial membrane potential as well as the
adenine nucleotide transferase concentration within the mitochondrial membrane. But the derivate 4-OHT is much less harmfull (CARDOSO ET AL. 2003).
According to LEONE ET AL. (2003) the mechanism of the regulation of the ERT2-Cas9 fusion protein should be the following: Without 4-OHT the protein remains in the cytoplasm, due to the shielding of the NLS by Hsp90s. When 4-OHT is applied to the cell it diffuses through the cell membrane and bind to ERT2 (Fig. 1 (1.) ). Because of this the heat shock proteins should separate from ERT2, which allows the fusion protein to enter the nucleus, where Cas9 (when in complex with crRNA and tracrRNA) can target - and regulate - the desired genes (Fig. 1 (2.) ).
Assembled plasmids
Due to unknown sterical side effects, 5 constructs with different NLS and ERT2 positions were assembled (see also Fig. 2):
First of all a NLS and an ERT2 were fused to both ends of the fusion protein, as a Cas9-GFP fusion protein with a NLS on both sides was shown by CONG
ET AL. (2013) to be localized exclusively in the nucleus within HEK cells. The from ERT2 in the inactive state acquired Hsp90s were thought to cover these
NLSs and therefore inhibit the nuclear import of the fusion protein. (pIG3005)
Secondly ERT2 was only fused to the N- or the C-terminal NLSs, because CONG ET AL. (2013) showed a difference in nuclear import efficiency due to the
position of the NLS. According to these studies the C-terminal NLS should have a higher impact on nuclear localization, but fusing ERT2 N-terminal leads to
a tighter hormone mediated control (MEIXLSPERGER ET AL. 2007). (pIG3006: ERT2 only C-terminal & pIG3007: ERT2 only N-terminal)
Thirdly constructs contain only one NLS (with ERT2). This should reduce the amount of uninduced nuclear import, while the size of the protein is lower
than pIG3005. (pIG3008: NLS and ERT2 only C-terminal & pIG3009: NLS and ERT2 only N-terminal)
At last for every NLS variation a control was assembled: Cas9-mCherry without a NLS (pIG3001), NLSs fused to both ends (pIG3002) and one NLS fused to
the C- (pIG3003) and to the N-terminus (pIG3004).
All planned plasmids could be successfully cloned by Gibson assembly.
Results
Influence of the NLS on nuclear import
Fluorescent pictures taken with the confocal microscope clearly show that pIG3001 is mostly localized in the cytoplasm, whereas with pIG3002 - 4 transfected cells display a much stronger red fluorescence in the nucleus. It seems that this nuclear localization is even stronger for pIG3003 & 4 (Fig. 3). This is confirmed by quantitative analysis of the mean fluorescence intensity: Fig. 5 shows a slight increase in the ratio of nuclear to cytoplasmic fluorescence from pIG3002 to 3 & 4, but within the error bars (standard deviation). The value of pIG3003 & 4 is more or less the same. The intensity ratio of pIG3002 is 14 fold, the intensity ratio of pIG3003 & 4 17 fold higher than the one of pIG3001.
Effects of 4-OHT induction on nuclear import
The red fluorescence of the Cas9-ERT2 fusion proteins is mostly localized in the nucleus both with 4-OHT and EtOH (negative control, as 4-OHT is dissolved in EtOH) treatment, a difference is not detectable, which is visible for pIG3007 & 9 in Fig. 4.
Quantitative analysis reveals a slight increase of the ratio of the mean nuclear red fluorescence intensity to the cytoplasmic upon 4-OHT treatment for pIG3005 (1,5 fold), pIG3007 (2 fold) and pIG3009 (1.3 fold), whereas this ratio drops upon 4-OHT treatment for pIG3006 (2 fold) and pIG3008 (1.6 fold), but for all within the standard deviation. All values range between the positive and the negative controls (Fig. 5).
Effects of 4-OHT on Cas9 functionality
As another aproach to regulate Cas9 activity, HEK cells were transfected with a constitutiv on SEAP plasmid, a RNA plasmid containing the tracrRNA
and a crRNA, that is complementary to a sequence at the beginning of the SEAP coding sequence, and the Cas9-ERT2 fusion constructs, which contain
two NLS. We hoped that the Hsp90s binding to ERT2 when there is no 4-OHT would sterically hinder Cas9 from binding to the target DNA.
Fig. 7 shows only very little differences in SEAP expression between hormone and ethanol treatment (in both directions) but 1.5 till 3 fold repression
when Cas9-ERT2 is transfected with a crRNA.
Toxicity of 4-OHT
In order to test if 4-OHT has some lethal side effects on HeLa cells, untransfected cells were treated with different 4-OHT concentrations (10 µM, 1 µM
and 0.1 µM), EtOH (1 % and 0.1 %) or none of them in two independent experiments for 18 respectively 24 h.
At the first experiment cells were observed by microscopy: All cells looked healthy, only the cells treated with 10 µM 4-OHT had a quite compact shape,
whereas all other had rather stretched shapes.
With the second experiment the number of dead cells in the supernatant were determined. The well with untreated cells contained the lowest number of
dead cells, the well with cells treated with 0.1 % EtOH the highest. Treatment with 4-OHT caused a little lower number of dead cells (Fig. 6).
Discussion
Influence of the NLS on nuclear import
The lack of a NLS leads to an almost exclusive cytoplasmic localization of the Cas9-mCherry fusion protein. The presence of a NLS increases the
nuclear import about 14 till 17 fold. Surprisingly one NLS seems to affect this stronger than two, but this difference is not significant due to the
overlapping standard deviations. The pictures may be misleading to this conclusion because of a different cell shape between pIG3002 and pIG3003/4
which is caused by a different cell density.
The observations that one NLS is at least as strong as two and that the N-terminal NLS is as strong as the C-terminal one do not agree with the results of
CONG ET AL. (2013). They showed that two NLSs lead to the strongest nuclear import, whereas a C-terminal NLS has a lower effect and a N-terminal NLS
causes almost no nuclear import. This divergence to our findings may be because CONG ET AL. used HEK cells (instead of HeLa cells) and a Cas9-GFP
fusion protein for their experiments. As GFP is derived from the fluorophor of Aequorea victoria (HEIM ET AL. 1994) and mCherry is a monomeric
derivative of the fluorophor of Discosoma sp. (SHANER ET AL. 2004), it should have a different tertiary structure, which may cause another
exposure of at least the C-terminal NLS.
Effects of 4-OHT induction
None of the 5 test constructs showed a big difference in subcellular localization after treatment with 4-OHT or EtOH; all values range within the
standard deviations (Fig. 5). So no fusion protein could be termed hormone inducible. But because of the high standard deviations, because there were
not so many cells on the pictures and because the regions where the mean fluorescence was measured had to be chosen personally, the analysis that
was performed is not really trustworthy. This would then be a explanation for a stronger nuclear localization with EtOH treatment.
The pictures of pIG3006 show the tendency of more nuclear localization after 4-OHT treatment (Fig. 5), whereas the quantitative analysis demonstrate the
opposite. The problem may be that with EtOH treatment there is a wide variety of cells with different fusion protein distributions, as indicated with the
high standard deviation.
Effects of 4-OHT on Cas9 functionality
Also the functionality of Cas9 could not be regulated with hormone induction: The observed repression between Cas9 with and without crRNA due to Cas9 binding to the coding sequencing of SEAP and therefore hindering the transcription is independent of hormone addition. With and without 4-OHT there is only a very slight difference; in most samples the repression is even stronger with ethanol addition than with tamoxifen (Fig. 9).
Toxicity of 4-OHT
4-OHT in concentrations below 1 µM does not seem to be toxic to HeLa cells. If it can be assumed that most dead cells will detach from the well, then
the toxicity assay we performed shows that the number of dead cells does not increase with 4-OHT treatment in compare to EtOH. It is more likely that
the solvent EtOH has a negative effect on HeLa cells. But to be sure about this, this assey would have to be repeated more times.
The change in shape cells show after treatment with 10 µM 4-OHT may also occur because of a high cell density.
As HeLa cells do not have an ER (KUMAR ET AL. 1987), only the weak effects of 4-OHT on mitochondria (CARDOSO ET AL. 2003) could harm the cells. But
for this the tested concentrations are most probably too low.
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