Abstract
Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.
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Acknowledgments
We thank Prof. Dr. Ralph Panstruga from RWTH Aachen, Germany, for providing MLO promoter sequence from Hordeum vulgare (cv. Ingrid).
We thank Prof. Reinhard Dammann, University Giessen, Institute for Genetics, for his project support with laboratory space and equipment.
This work was supported in the program PLANT 2030 (project āBarEpiEditā) funded by the Federal Ministry of Education and Research, Germany (BMBF), and due to the LGFG-scholarship of the Ministry of Science, Research and the Arts Baden-WĆ¼rttemberg, Germany. Additionally, we are thankful for the invitation for contributing our method in the lab protocol series Methods in Molecular Biology published by Springer Nature.
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Hinrichs, AK., Koch, A., Richter, A. (2024). Dual-Luciferase Reporter Assay for Prescreening CRISPR (d)Cas9-Mediated Epigenetic Editing on a Plant Promoter Using Human Cells. In: Maghuly, F. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 2788. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3782-1_16
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DOI: https://doi.org/10.1007/978-1-0716-3782-1_16
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3781-4
Online ISBN: 978-1-0716-3782-1
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