Difference between revisions of "Part:BBa K1150024"

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uniCAS Histone Modifier (CMV promoter)

This device combines the dCas9 protein with the set-domain of the murine histone methyltransferase G9a. dCas9 enables not only specific, but also multiple targeting of any DNA sequence(s) of interest. Hence, coupling of dCas9 to the effector G9a enables specific methylation of histone H3 lysin 9 at any desired and accessible locus. These G9a induced histone modifications render open gene loci to an transcriptionally inactive state. [1]

To ensure localization to its site of action the dCas9, fused to G9a via a short linker, was flanked by two nuclear localization signals (NLS). Additionally, to enable detection of the expressed protein, it was tagged by an HA-epitope. utilizing the strong CMV promoter in combination with the BGH terminator this device allows for constitutive, mammalian expression.

Figure 1.: Complete overview on CMV:dCas9-G9a with all features.

Usage and Biology

H3K9 methylation is a hallmark of repressed transcriptional states. [2] Upon close contact the murine G9a-set domain transfers methyl groups to H3K9leading to transcriptional repression. [3] The dCAS9 protein can simultaneously be targeted to several DNA loci as it interacts with small RNAs thereby forming a complex that will interact with complementary DNA strands. It origins from the bacterial adaptive immune system of Streptococcus pyogenes called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting. The team Freiburg 2013 combined these two elements to create a transcriptional repressor that can be targeted to any desired locus of interest.
This approach offers new possibilities for applied as well as for fundamental research, such as tissue engineering, epigenetics and cancer research.

Functional tests

Expression

To ensure protein expression of the fusion protein HEK293T cells were seeded into 6-wells at a density of 150.000 cells per well. 24 hours later they were transfected with this device.
2 days post transfection the protein expression was evaluated by Western blot analysis of total cell lysates. Figure 2 demonstrates successful expression of the dCas9-G9a fusion protein, by detection the HA-tag.

Figure 2.: HA-tag fused to different dCas9-fusion proteins encoded on the RFC25 pSB1C3 backbone.

Proof of Function

After knowing, that the protein is expressed functional tests were performed. As stated before, when targeted to an open, endogenous locus we expect change of the epigenetic state to repression. To test this, the endogenous VEGF-A locus in HEK293-T cells was chosen, as it is well-characterized and open in HEK293T cells. Additionally it can easily be measured by ELISA analysis.
Cells were seeded into 24-well format and transfected with the plasmid and different crRNAs, coded by derviative plasmids of the RNAimer, that code for loci on the VEGF gene. These were: -8, -573, +434, -475 and -573/+434. As an off-target control the plasmid coding for the crRNA EMX was used. One control does not contain any target RNA. Every target site was tested in parallel with only CMV:dCas9, to differentiate the action of G9a against the CRISPRi effect. As an internal standard a constitutive SEAP reporter was used. After 24 hours, VEGF levels and SEAP levels were measured and the ratio was calculated. Error bars represent standard deviation.

Figure 3.: dCas9-G9a targetting of endogenous VEGF-A locus in HEK cells.


ELISA values, normalized to internal standard SEAP. It is clearly visible that for some loci a repressive effect is detectable, roughly 50%. Controls did not displays any off-target effects. Combining two targets sites did not show any effect, in contrary it seems to decrease the effect.
It is clearly visible, that a repressive effect is there, when dCas9-G9a is targeted to loci in the VEGF gene. The loci have different effects and combining two on one plasmid does not seem to have a good repression. The off-target controls do not display a repressive effect.
In summary, the CMV:dCas9-G9a is an effective repressor of endogenous gene expression, by modulating the chromatin structure of the targeted locus. this offers application in fundamental research as well as in medical science.

[1]Wolffe, A., et al. (1999). Epigenetics: Regulation Through Repression. Science 286169, 481.
[2]Snowden, A., et al. (2002). Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo. Current Biology 12, 2159-2166.
[3] Lee, D., et al. (2006). Histone 3 Lysine 9 Methyltransferase G9a Is a Transcriptional Coactivator for Nuclear Receptors. Journal of Biological Chemistry 281, 8476-8485.

Sequence and Features