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Team:Freiburg/Project/induction
From 2013.igem.org
Effector Control
We engineered the CRISPR/Cas9 system that is relying on a protein-RNA-DNA interaction to generate a DNA bidning protein. For this we used a catalytically inactive Cas9, the dCas9. After generating a series of effectors using the CRISPR/Cas9 system, we aimed to control our system via external stimulation. We developed two approaches for induction, either on hormone stimulus or on light stimulus.
To test this system we performed functional tests, using the RNAimer. This plasmid codes for the RNAs that are necessary for the binding ability of dCas9.
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 [1], [2]. However these systems often lack reversibility, exact spatial control and show toxic and pleiotropic side effects [3], [4]. These problems can be solved using for instance optogenetic tools to control gene expression [5], [6] or other desired interactions [7] e.g. activation of protein splicing [8]. 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 [9] to the practical use in synthetic biology [10] only few years passed. The recently emerged CRISPR/Cas system established in mammalian cells allowed first applications in 2011 [11]. The aim of this study is to connect both fields to form a new system for the use in mammalian cells. The combination of dCas9 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. 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 [12].
Our universal toolkit will allow you to use the full spectre of light as induction signal. All proteins are derived from Arabidopsis thaliana. Phytochrome B is responsible for red-light induction. Cryptochrome 2 is responsible for blue-light induction and the UV-light receptor UVR8 is responsible for induction of UV-B light.
Effector Control by 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 125 kDa that is predominantly located in the cytoplasma. Illumination with red light (660nm wavelength) leads to binding of Phytochrome Interaction 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 dCas9 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. PhyB is a protein with a molecular mass of 125k Da that is predominantly located in the cytoplasma. Its structure can be roughly divided in two parts: The N-terminal part, regulatory and photosensory, and the C-terminal part, regulatory. The N-terminal part is covalently bound to phytochromibilin. Phytochromibilin is a linear tetrapyrrol that undergoes conformational changes upon radiation of either red- or far red-light when bound to the phytochrome moiety. This conformational change is responsible for the photosensory properties of PhyB [13], [14].
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| Figure 1: Plasmid-cards of red-light effector-control plasmids For gene-activation via red-light stimulus, we constructed a fusion of dCas9 linked to PIF6 for exact gene targeting (BBa_K1150025) . To enable gene activation, we linked PhyB to a VP16-activation domain (BBa_K1150026) . Both constructs contain a Nuclear Localisation Sequence, nessesary for usage in mammalian cells. Additionally the the expression of dCas9-PIF6 can be quantified using the attached HA-tag. |
The dCas9 protein has built a complex with cr- and tracr RNA, additionaly it is covalently linked to PIF6. After expression, it will bind to its target locus. A red-light stimulus is recruiting the VP16 protein linked to PhyB and thereby initiating expression of a target gene. Red light does not carry any pleiotropic or toxic side effects. Red light additionally has no effects on other light-absorbing particles, that may be disrupted by illumination. The dCas9-protein is able to target even multiple gene-loci with only one transfection. Targeting one locus with multiple cr-RNAs may enhance the desired effect; either inhibition or induction of target genes.
| Figure 2: Mechanism of red-light induction The fusion protein consisting of Cas9 and PIF6 binds to its target locus. Illumination with red-light leads to recruition of the activating fusion-protein PhyB-VP16. |
Experiments
The Experiments for the induction with red light were executed in the following pattern:
Day 0: Seeding of Cells. 65,000 Cells were seeded into a 24-well plate.
Day 1: Transfection of Cells. The Transfection was executed using PEI at a PEI/DNA-ratio of 3:1. Medium was changed 5h post transfection.
Day 2: 1h prior to illumination with red light, 660nm, or far-red light, 740nm, (20uE for both), the medium was renewed with fresh media containing 15uM phycocyanobilin
Day 3: Illumination (48h total).
Day 4: The desired assay (SEAP-measurement) can be performed.
Results
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Figure 3: SEAP levels for HEK293T-cells. The HEK293T-cells were treated as described in the "Experiments" section above. The bars on the left side show SEAP levels of the negative control that was transfected without the targeting plasmid (BBa_K1150034, RNAimer). The bars on the right side show SEAP levels of constructs transfected with target plasmid. Red bars show cell-samples illuminated with activating red-light, white bars show samples illuminated with inactivating far-red light. Error bars show standard deviation of biological triplicates. An increasing SEAP-level after illumination can be observed if target plasmid is present. |
The negative controls indicate that the used reporter plasmid containing the SEAP coding sequence shows a high leaky SEAP expression. Due to this high background level, the induced activation by of the light-system is only slightly obserrvable.
In order to improve the induction further experiments need to be performed, e.g. usage of different cell-lines, other reporter systems, more target sites and different effector proteins (VP64, p65).
Effector control by UVB-light stimulus
Another light system that we engineered is based on the induction by ultraviolet B light (UVB) with a wavelength of 311 nm. The main components are the UV resistance locus 8 (UVR8) and the WD40 domain of COP1 as COP1 stands for E3-ubiquitin ligase Constitutively Photomorphogenic 1 [15], [16]. Both proteins derive from A. thaliana. UVR8 is a UVB light-responsive receptor that stays in his closed configuration without light stimulus. When exposed to UVB light the configuration changes to its open state and recruits COP1 [6].
By combining our dCas9 protein and effector domains with these proteins we engineered a UVB light inducible gene regulation system.
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| Figure 4: Plasmid-cards of UV-b light effector-control plasmids For gene-activation via UV-b light stimulus, we constructed a fusion of dCas9 linked to UVR8 for exact gene targeting (BBa_K1150029) . To enable gene activation, we linked COP1 to a VP16-activation domain (BBa_K1150030) . Both constructs contain a Nuclear Localisation Sequence, nessesary for usage in mammalian cells. Additionally the expression of dCas9-UVR8 can be quantified using the attached HA-tag. |
The core domain of UVR8 was fused to our dCas9 protein. The other transcription factor consists of COP1 fused to the transactivation domain VP16 or repressor domain KRAB. When crRNA and tracrRNA bind to dCas9, the protein is able to bind complementary DNA. If the system is exposed to UVB light (311 nm) the UVR8 receptor changes its configuration and recruits COP1 with for example its fused transactivation domain VP16 which starts gene expression. Subsequently the gene of interest is activated.
| Figure 5: Mechanism of UV-b light induction The fusion protein consisting of Cas9 and UVR8 binds to its target locus. Illumination with UV-b light leads to recruition of the activating fusion-protein COP1-VP16. |
Experiments
The Experiments for the induction with UV-B light were executed in the following pattern:
Day 0: Seeding of Cells. 65,000 Cells were seeded into a 24-well plate.
Day 1: Transfection of Cells. The Transfection was executed using PEI at a PEI/DNA-ratio of 3:1. Medium was changed 5h post transfection.
Day 2: Illumination with UV-B light: 5uE for 24h
Day 3: The desired assay can be performed.
Results
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Figure 6:SEAP levels for HEK293T-cells. The HEK293T-cells were treated as described in the "Experiments" section above. The bars on the left side show SEAP levels of the negative control that was transfected without the targeting plasmid (BBa_K1150034, RNAimer). The bars on the right side show SEAP levels of constructs transfected with target plasmid containing 3 different target sites. Violet bars show cell-samples illuminated with activating UV-b light, white bars show samples that were kept in darkness. Error bars show standard deviation of biological triplicates. An increasing SEAP-level after illumination can be observed, if target plasmid is present. |
The results show a almost 2-fold induction after illumination. The negative controls without Cas9 target sites indicate that the used reporter plasmid has a highly leaking SEAP expression in HEK293T cells. Due to this high background level, the induced activation by of the light-system is only slightly obserrvable.
In order to improve the induction further experiments need to be performed, e.g. usage of different cell-lines, other reporter systems, more target sites and different effector proteins (VP64, p65). Other cell lines may also tolerate longer illumination times.
Effector control by blue-light stimulus
The third light system we engeneered is the blue light mediated induction. The system we used is based on dimerization of cryptochrome 2 (Cry2), which is located in cytosol and the calcium and integrin binding protein 1 (CIB1), which is located in the nucleus.
Cryptochrome 2 binds flavin and pterin on its conserved N-terminal photolyase homologe region. This interaction leads to the blue-light responsiveness of Cry2.
These two components were first found in Arabidopsis thaliana, but are also expressed in mammalian cells. So the blue light system requires no exogeneous chromophore. [17], [18].
For our system we fused Cry2 to the Cas9 and CIB1 to different effector domains. By fusing VP16 and KRAB to CIB1 we build a blue light mediated possibility to activate or repress genes.
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| Figure 7: Plasmid-cards of blue-light effector-control plasmids For gene-activation via UV-b light stimulus, we constructed a fusion of dCas9 linked to CRY2 for exact gene targeting. To enable gene activation, we linked CIB1 to a VP16-activation domain. Both constructs contain a Nuclear Localisation Sequence, nessesary for usage in mammalian cells. Additionally the expression of dCas9-CRY2 can be quantified using the attached HA-tag. |
When crRNA and tracrRNA bind to dCas9, the protein is able to bind complementary DNA. If the system is exposed to blue light (465 nm) the Cry2 receptor recruits CIB with its fused transactivation domain VP16 which starts gene expression. Therefore the gene of interest is activated.
Experiments
Day 0: Seeding Cells: 65,000 CHO-cells were seeded into 24-well plates.
Day 1: The Transfection was executed using PEI at a PEI/DNA-ratio of 3:1. Medium was changed 5h post transfection.
Day 2: Media change before light treatment, Illumination with blue light (465nm): 5uE for 24h
Day 3: The desired assay can be performed.
| Figure 8: Mechanism of blue-light induction The fusion protein consisting of Cas9 and CRY2 binds to its target locus. Illumination with blue light leads to recruition of the activating fusion-protein CIB1-VP16. |
Results
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Figure 9: SEAP levels for NIH/3T3-cells. The NIH/3T3-cells were treated as described in the "Experiments" section above. The bars on the left side show SEAP levels of the negative control that was transfected without the targeting plasmid containing 4 different target sites (BBa_K1150034, RNAimer). The bars on the right side show SEAP levels of constructs transfected with target plasmid. Blue bars show cell-samples illuminated with activating blue light, white bars show samples that were kept in darkness. Error bars show standard deviation of biological triplicates. An increasing SEAP-level after illumination can be observed, if target plasmid is present. |
The results show a almost 5-fold induction after illumination. In general we could only observe lower absolute SEAP levels, but nevertheless a clear induction. This might be due to the lower transfection efficiency of NIH/3T3 cells and overall lower expression levels
The background SEAP level in this cell line was, in contrast to the HEK293T cells, quite low.
References
Hormone
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) [1].
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 [2]. For the glucocorticoid
receptor (GR) this interaction is crucial for nuclear import [3] . Even within the nucleus most steroid receptors are in complex with Hsp90
[4]. 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 [2].
Tamoxifen is a competitive inhibitor of ER. For that reason it is widely used for treatment of breast cancer [5]. In the body of higher
vertebrates Tamoxifen becomes metabolized to 4-OHT [6]. This form is more potent in inhibiting the ER which leads to a stronger
repression of estrogen-dependent cell growth [7] .
In 1993 Danielian et al. [8] mutated the murine ER, so that it is no more sensitive to estrogen, but becomes active after binding of tamoxifen. Littlewood et al. [9] 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 [10]. For the
regulation 4-OHT is preferred to tamoxifen because it yields a higher effect with lower concentrations [11].
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 [7].
According to Leone et al. [12] 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.) ).
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| Fig. 1: Proposed mechanism Without (Hydroxy)tamoxifen in the cell the fusion protein (Cas9, NLS & ERT2) is in complex with Hsp90 chaperones, which should cover the NLS. After tamoxifen binding (1) to the HBD, the chaperones separate and the fusion protein will be imported into the nucleus (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. [13] 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. [13] 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 [14]. (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.
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| Fig. 2: Assembled fusion proteins All fusion proteins used for the hormone induction are pictured with all their essential parts. pIG3001 - 4: controls; pIG3005 - 9: test constructs; HA: HA tag; ERT2: hormone receptor domain; NLS: nuclear localization sequence. |
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).
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| Fig. 3: Fluorescence pictures of HeLa cells expressing the control constructs Pictures were taken with a confocal fluorescence microscope. mCherry: fluorophor (part of the Cas9-ERT2 fusion protein); DRAQ5: DNA staining; pIG3001: Cas9-ERT2 fusion protein without a NLS; pIG3002: Cas9-ERT2 fusion protein with a NLS on both ends; pIG3003: Cas9-ERT2 fusion protein with a C-terminal NLS; pIG3004: Cas9-ERT2 fusion protein with a N-terminal NLS. |
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.
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| Fig. 4: Fluorescence pictures of HeLa cells expressing pIG3006 & 8 (ERT2 at the C-terminus) Pictures taken with a confocal fluorescence microscope were chosen as an example for the look of the cell expression the Cas9-ERT2 fusion proteins. mCherry: fluorophor (part of the Cas9-ERT2 fusion protein); DRAQ5: DNA staining; pIG3006: Cas9-ERT2 fusion protein with a NLS on both ends and a ERT2 at the C-terminus; pIG3008: Cas9-ERT2 fusion protein with a C-terminal NLS and ERT2. The very bright, round red shapes are mostly dead cells. |
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).
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| Fig. 5: Ratio of the mean intensity of nuclear and cytoplasmic fluorescence For each bar the mean intensities of nuclear and cytoplasmic red fluorescence of at least 7 cells on one picture taken by the confocal microscope were determined using ImageJ. This was done by selecting a region in the nucleus and in the cytoplasm of each cell. The figure shows the ration of nuclear to cytoplasmic fluorescence (background subtracted) with the standard deviation. The grey bars represent the controls (no treatment): pIG3001 = negative control (no NLS); pIG3002 - 4 = positive controls (one or two NLSs, respectively). The blue bars represent the test constructs with either 0.1 % EtOH or 1 µM 4-OHT treatment over night. |
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. 6 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.
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| Fig. 6: Repression of SEAP expression The SEAP activities of technical triplicates were measured after the addition of a certain amount of pNPP (substrate of SEAP). The bars represent the mean with standard deviation. red: controls without Cas9-ERT fusion proteins; blue: tests with Cas9-ERT fusion proteins; 1/5: cotransfection of a RNA plasmid containing a crRNA targeting the SEAP coding region at the first 1/5; 1/10: cotransfection of a RNA plasmid containing a crRNA targeting the SEAP coding region at the first 1/10; -/-: no RNA plasmid transfected; 4-OHT: treatment with 4-OHT; EtOH: treatment with EtOH. |
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. 7).
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| Fig. 7: Number of dead cells in supernatant of HeLa cell culture after different treatment Cells were treated with 4-OHT or EtOH as shown in the figure for 18 h. Cells within the removed supernatant and 0.5 ml PBS of a washing step were counted and dead cells were calculated. 1 µM = 1 µM 4-OHT; 0.1 µM = 0.1 µM 4-OHT. |
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. [13]. 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 [15] and mCherry is a monomeric
derivative of the fluorophor of Discosoma sp. [16], 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. 7).
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 [1], only the weak effects of 4-OHT on mitochondria [7] could harm the cells. But for this the tested concentrations are most probably too low.
References
