Versatile molecular tools enabling customizable DNA methylation editing in plants [ATAC-seq]
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE282160 | GEO | 2025/11/10
REPOSITORIES: GEO
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Project description:Tools to edit DNA methylation in a targeted manner are vital for clarifying the causal relationships between DNA methylation and its function, as well as for plant breeding and gene therapy; non-specific tools that induce genome-wide DNA methylation changes are valuable for the creation of novel epialleles and performing large-scale screens. Here, by constructing dCas9 fusions to a panel of effectors and cofactors, we uncovered a range of tools for editing DNA methylation, including five tools for DNA methylation and six tools for DNA demethylation. Our tools show diversity of performance in terms of specificity and efficiency, offering either the capacity to target editing DNA methylation or the ability to edit DNA methylation at the genome-wide level. Remarkably, DNA methylation edited by these tools was inherited in the absence of transgene. These versatile tools pave the way for diverse applications in DNA methylation editing and hold promise for advancing plant epigenetic breeding.
2025-11-10 | GSE282162 | GEO
Project description:Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid-editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.
2022-02-03 | GSE195977 | GEO
Project description:Epialleles are meiotically inherited variations in expression states that are not linked to changes in DNA sequence. Although they are well documented to persist in plant genomes, their molecular origins are unknown. Here, we show using a variety of mutant and experimental populations that epialleles in Arabidopsis thaliana result from feedback regulation of pathways that primarily function to maintain DNA methylation at heterochromatin. Perturbations to maintenance of heterochromatin methylation leads to feedback regulation of DNA methylation in genes, with a preference for genes with pre-existing DNA methylation. Using epigenetic recombinant inbred lines (epiRIL), we show that epiallelic variation is enriched in euchromatin, yet, associated with QTL primarily located in heterochromatin. Mapping three-dimensional chromatin contacts reveals that genes that are hotspots for epiallelic variation have increased contact frequencies with regions possessing H3K9me2. Altogether, these data show that feedback regulation of pathways that evolved to maintain heterochromatin silencing leads to the origins of spontaneous epialleles.
2021-11-01 | GSE171157 | GEO
Project description:Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice demonstrating their wide utility for functional studies of epigenetic regulation.
2016-09-23 | GSE83890 | GEO
Project description:Genes and transposons can exist in variable DNA methylation states with potentially differential transcription. However, how these epialleles emerge is poorly understood. Here we show that crossing a hypomethylated plant with its normally methylated wild-type counterpart results already in the F1 generation in widespread changes in DNA methylation and transcription patterns. Novel non-parental and heritable epialleles arise at many genic loci, including a locus that itself controls DNA methylation patterns, but with most of the changes affecting pericentromeric transposons. While a subset of transposons show immediate resilencing, a large number displays decreased DNA methylation, which is associated with de novo or enhanced transcriptional activation. This can translate into transposon mobilization in the progeny. Our findings reveal that the combination of distinct epigenomes can be viewed as an epigenomic shock, which is characterized by a round of epigenetic variation creating novel patterns of gene and TE regulation.
2015-07-20 | E-MTAB-4483 | biostudies-arrayexpress
Project description:DNA methylation is important for silencing genes and transposable elements. Changes in DNA methylation can be heritable and thus, the loss or gain of methylation can lead to the formation of stable epialleles. A well characterized example of a stable epiallele in plants is fwa-4, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing upregulation of FWA and a heritable late flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the Human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA upregulation, and a heritable late flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCAS9 based targeted demethylation system using the TET1cd and a modified SunTag system and demonstrate its ability to target demethylation at and activate the expression of FWA and CACTA1. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.
2018-02-18 | GSE109115 | GEO
Project description:Huge efforts are made to engineer safe and efficient genome editing tools. An alternative might be the harnessing of ADAR-mediated RNA editing. We now present the engineering of chemically optimized antisense oligonucleotides that recruit endogenous human ADARs to edit endogenous transcripts in a simple and programmable way, an approach we refer to as RESTORE. Notably, RESTORE was markedly precise, and there was no evidence for perturbation of the natural editing homeostasis. We applied RESTORE to a panel of standard human cell lines, but also to several human primary cells including hepatocytes. In contrast to other RNA and DNA editing strategies, this approach requires only the administration of an oligonucleotide, circumvents the ectopic expression of proteins, and thus represents an attractive platform for drug development. In this respect we have shown the repair of the PiZZ mutation causing α1-antitrypsin deficiency and the editing of phosphotyrosine 701 in STAT1.
2018-12-01 | GSE121573 | GEO
Project description:DNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of the tools at a genome wide level which revealed consistent off-target binding events and DNA methylation deposition throughout the genome, limiting the capacity of these tools to generate unambiguous determination of the functional consequences of DNA methylation. To address this issue, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA-methylation. dC9Sun-D3A is tunable, specific and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the least amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.
2018-06-05 | GSE107607 | GEO
Project description:The transgenerational stability of DNA methylation changes is important in setting up genomic DNA methylation patterns and in the formation and transmission of epialleles. It is generally assumed that DNA methylation changes at genomic regions targeted by the de novo, RNA-directed DNA methylation (RdDM) pathway are unstable. Here, we show that RdDM targets in Arabidopsis can be classified into two groups based on their transgenerational epiallele stability following restoration of NRPD1 function in nrpd1 mutant plants: remethylable loci and non-remethylable loci. Compared to the remethylable loci, non-remethylable ones contain higher levels of the euchromatic marks H3K4me3 and H3K18ac, which interferes with the recruitment of the RdDM molecular machinery and helps to recruit the DNA demethylase ROS1 to antagonize RdDM, respectively. Using targeted de-methylation by CRISPR/dCas9-TET1, we demonstrate that mCG and mCHG are memory marks required for targeting the RdDM machinery to remethylable loci. Our results show that histone and DNA methylation marks are critical in determining the capacity of RdDM target loci to form stable epialleles, and contribute to understanding the formation and transmission of epialleles.
2020-04-17 | GSE140566 | GEO
Project description:Magnetic miniature robotic systems have attracted broad research interest because of their precise maneuverability in confined spaces and adaptability to diverse environments, holding significant promise for applications in both industrial infrastructures and biomedical fields. However, the predominant construction methodology involves the preprogramming of magnetic components into the system's structure. While this approach allows for intricate shape transformations, it exhibits limited flexibility in terms of reconfiguration and presents challenges when adapting to diverse materials, combining, and decoupling multiple functionalities. Here, we propose a construction strategy that facilitates the on-demand assembly of magnetic components, integrating ferrofluid droplets with the system's structural body. This approach enables the creation of complex solid-droplet robotic systems across a spectrum of length scales, ranging from 0.8 mm to 1.5 cm. It offers a diverse selection of materials and structural configurations, akin to assembling components like building blocks, thus allowing for the seamless integration of various functionalities. Moreover, it incorporates decoupling mechanisms to enable selective control over multiple functions, leveraging the fluidity, fission/fusion, and magneto-responsiveness properties inherent in the ferrofluid. Various solid-droplet systems have validated the feasibility of this strategy. This study advances the complexity and functionality achievable in small-scale magnetic robots, augmenting their potential for future biomedical and other applications.
| S-EPMC11317579 | biostudies-literature