"TRADES": Targeted RNA demethylation by Suntag system
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PROVIDER: PRJNA586772 | ENA |
REPOSITORIES: ENA
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Project description:DNA methylation is a stable and heritable epigenetic mark, and it plays an important role in regulation of gene expression and transposon silencing. Here we developed a CRISPR/dCpf1-based targeted demethylation system using the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and a SunTag system. The SunTag-dCpf1-TET1cd system is able to achieve targeted DNA demethylation and ur-regulate gene expression when guided to the FWA or CACTA1 loci in Arabidopsis thaliana. Our study provides tools for targeted removal of DNA cytosine methylation, and activation of protein-coding genes or transposons expression.
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Project description:CRISPR-based epigenome editing was recently used to activate gene expression through direct transcriptional activation or site-specific DNA demethylation. Viral delivery of guide RNAs for these purposes remains to be developed. Furthermore, currently available viral delivery tools for genome editing show meager rates of heritability. Here, we have developed a tobacco rattle virus (TRV)-based guide RNA delivery system for both transcriptional activation and targeted DNA demethylation. To promote heritable epigenome editing specifically within plant meristems and the germline, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome, thus facilitating cell-to-cell movement of the RNA in plants. We achieved up to ~8% heritability of the induced phenotype in the progeny of virus-inoculated plants and 25% in the following generation, indicating high rates of heritability for targeted DNA demethylation. Thus, TRV delivery, in combination with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.
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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.
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Project description:Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells (iPSCs) is not known. Here we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes. Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires AID-mediated downregulation of UHRF1 protein and abolishing demethylation leaves thousands of hypermethylated regions in the iPSCs genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network. Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity.
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Project description:DNA methylation has been utilized for target gene silencing in plants, however it’s not well-understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain-of-function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression through DNA methylation, histone H3K27me3 deposition, H3K4me3 demethylation, histone deacetylation, inhibition of RNA Polymerase II transcription elongation or Ser-5 dephosphorylation. These proteins also silenced many other genes with different efficacy, and a machine learning model could accurately predict the efficacy of each silencer based on various chromatin features of the target loci. Furthermore, some proteins were also able to target gene silencing when used in a dCas9-SunTag system. These results provide a more comprehensive understanding of epigenetic regulatory pathways in plants and provide an armament of tools for targeted gene manipulation.
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Project description:Deficiency of the N6-methyladenosine (m6A) methyltransferase complex results in global reduction of m6A abundance and defective cell development in embryonic stem cells (ESCs). However, it’s unclear whether regional m6A methylation would affect cell fate decisions due to the inability to modulate individual m6A modification in ESCs with precise temporal control. Here, we develop a targeted RNA m6A erasure (TRME) system to achieve site-specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline-inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6A site of mRNA and increase mRNA stability with limited off-target effects. We further demonstrate that temporal m6A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.
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