Abstracts
Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells
Anja R. Köhler
University of Stuttgart Germany, Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, Allmandring 31, 70569 Stuttgart [DE], anja.koehler@ibtb.uni-stuttgart.de
Author(s):
Anja R. Köhler, Pavel Bashtrykov, Albert Jeltsch
The precise spatio-temporal expression of genes in mammalian cells is regulated by epigenomic modifications on DNA and histones. To understand the dynamic changes in these modifications that occur during differentiation and disease onset in live cells, we developed epigenetic sensors to visualize locus-specific epigenome modifications via fluorescence microscopy [1]. The Bimolecular Anchor Detector (BiAD) technology combines an sgRNA/dCas9 complex as a programmable DNA-binding anchor with chromatin reader domains binding specific epigenome modifications as detector modules. Both modules are fused to complementary parts of a split fluorophore, reconstituting a full fluorophore upon binding in close proximity. To further enhance the sensors’ sensitivity towards a broader range of endogenous target loci, we recruited a second, full fluorophore to the sgRNA to mark the genomic locus of interest [2]. This novel dual-color readout, combined with an expanded detector module palette, enabled the quantitative analysis of various key epigenome modifications. We showcased applications of the dual-color BiAD technology in development and disease research by detecting dynamic changes in locus-specific DNA methylation upon inhibitor treatment and dissecting the enzymatic network responsible for the deposition of a critical repressive histone modification at a specific locus on the female inactive X chromosome.
[1] Lungu et al., 2017, Nat. Comm.
[2] Koehler et al., 2024, Cell Reports Methods