Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.
Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.
Seven bis(2-picolyl)amine (bpa) and five iminodiacetamide (imda) ligands were prepared with different modifications in their side chain structure. The coordination properties of the ligands (L) were influenced by changes in...
Genome editing tools, such as TALEN (transcription activator-like effector nuclease) or CRISPR-Cas9 (CRISPR-associated protein-9 nuclease) systems, enable functional studies by targeted gene knockout. They introduce double-stranded breaks (DSBs) into a DNA molecule in a sequence-specific manner, thereby stimulating the error-prone non-homologous end joining repair mechanism, leading to probable gene inactivation when the coding sequence is targeted. Vectors for expression of TALEN and Cas9-based constructs targeting the human IL6ST and HNF1A genes were assembled and tested for their ability to introduce DSBs when transfected into cultured cells using the luciferase assay. The Cas9-based construct targeting the IL6ST gene was shown to be active, while the two TALEN-based constructs did not introduce DSBs above background level. Both the TALEN and the CRISPR-Cas9 constructs targeting the HNF1A gene were found to be active, with the TALEN showing higher activity in a dose-dependent manner. The constructed genome-editing tools can be used for functional analysis of the putative role of HNF1A and IL6ST genes in IgG glycosylation, as shown previously by genome wide association studies.
The binding interactions of six ligands, neutral and monocationic asymmetric monomethine cyanine dyes comprising benzoselenazolyl moiety with duplex DNA and RNA and G-quadruplex structures were evaluated using fluorescence, UV/Vis (thermal melting) and circular dichroism (CD) spectroscopy. The main objective was to assess the impact of different substituents (methyl vs. sulfopropyl vs. thiopropyl/thioethyl) on the nitrogen atom of the benzothiazolyl chromophore on various nucleic acid structures. The monomethine cyanine dyes with methyl substituents showed a 100-fold selectivity for G-quadruplex versus duplex DNA. Study results indicate that cyanines bind with G-quadruplex via end π-π stacking interactions and possible additional interactions with nucleobases/phosphate backbone of grooves or loop bases. Cyanine with thioethyl substituent distinguishes duplex DNA and RNA and G-quadruplex structures by distinctly varying ICD signals. Furthermore, cell viability assay reveals the submicromolar activity of cyanines with methyl substituents against all tested human cancer cell lines. Confocal microscopy analysis shows preferential accumulation of cyanines with sulfopropyl and thioethyl substituents in mitochondria and indicates localization of cyanines with methyl in nucleus, particularly nucleolus. This confirms the potential of examined cyanines as theranostic agents, possessing both fluorescent properties and cell viability inhibitory effect.
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