Part:BBa_K5195001
Kruppel Associated Box (KRAB) Repressor
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Description
Base pairs: 195
Origin: pHAGE EF1α dCas9-KRAB, synthetic
Properties: Serves as an inhibitor to the binding of DUX4-FL
Usage and Biology
We binded DUX4-DBD to the repressor sequence of KRAB, and transfected this new construct in HEK 293T cells to observe and quantify reporter expression. With decreased fluorescent reporter activity, DUX4-DBD–KRAB serves as another promising approach to further inhibit DUX4-FL.
The Krüppel-associated box (KRAB) has homology to about one-third of human zinc finger proteins found in humans. With such a characteristic, it makes it highly favorable to bind to another endogenous human protein to regulate transcription.[1]
With FSHD patients losing the ability to repress DUX4-FL, encoded by D4Z4 chromosomal repeats, researchers aim to find regulators of this region. To correct this epigenetic limitation, dCas9-KRAB was utilized as a transcriptional inhibitor to further reduce DUX4-FL expression. The dCas9 protein is catalytically inactive, and being bound to the KRAB repressor, it has been shown to demonstrate properties that act as an effective transcriptional repressor. This is the case when dCas9-KRAB is recruited close to the transcription start site of genes of interest.[2]
Design
To integrate the fragment into our DBD backbone, we added overhangs to the sequence via PCR. We considered making a shorter isoform of DUX4-DBD (157 amino acids, which includes only the two homeodomains) and attaching a KRAB repressor to this sequence. Our team only cloned the original design since DBD 157 proved to not have much change to the competitive properties of our initial DBD.
Characterization
Part characterization for the KRAB domain was performed using its composite part BBa__K5195011 (KRAB fused to DUX4-DBD and linked to a mNeonGreen fluorophore). Please refer to that part page for more details.
References
[1] Margolin, J. F., Friedman, J., Meyer, E. K.-H., Vissing, H., & Thiesen, H.-J. (1994, January 31). Kruppel-associated boxes are potent transcriptional repression domains. National Library of Medicine | National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC43815/pdf/pnas01132-0416.pdf
[2] Himeda, C. L., Jones, T. I., Virbasius, C.-M., Zhu, L. J., Green, M. R., & Jones, P. L. (2018). Identification of epigenetic regulators of DUX4-FL for targeted therapy of facioscapulohumeral muscular dystrophy. Molecular Therapy, 26(7), 1797–1807. https://doi.org/10.1016/j.ymthe.2018.04.019
[3] Kearns, N. A., Genga, R. M., Enuameh, M. S., Garber, M., Wolfe, S. A., & Maehr, R. (2014). Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development, 141(1), 219–223. https://doi.org/10.1242/dev.103341
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