Epigenetic Editing: On the Verge of Reprogramming Gene Expression at Will

Cano-Rodríguez, David; Rots, Marianne G.

doi:10.1007/s40142-016-0104-3

Cited by 54 publications

(29 citation statements)

References 97 publications

(85 reference statements)

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“…Fusion of epigenetic effector domains with ZFs resulted in the same magnitude of silencing as the ZF-SKD fusions, indicating that our approach worked as we aimed for. Furthermore, targeted DNA methylation or histone methylation has the advantage that its effect has the potential to be permanent (4,28,31), albeit the stability and heritability of epigenetic reported (1,19,31). Here, we provide indications that targeting epigenetic effector domains to SPDEF has the ability to promote sustained gene expression reprogramming.…”

Section: Discussionmentioning

confidence: 80%

“…The SKD domain and the DNMT3A3L catalytic domain and its catalytic mutant (29) were subcloned by amplifying with primers containing MluI and NotI overhangs. Cloning of guide RNAs (gRNA) was achieved as previously described (4). Briefly, pairs of DNA oligonucleotides encoding 20 nucleotide gRNA targeting sequences were annealed together to create double-stranded DNA fragments with 4-bp overhangs.…”

Section: Plasmids Constructsmentioning

confidence: 99%

“…Epigenetic editing is a novel approach to modulate epigenetic states locally by targeting an epigenetic enzyme to the locus of interest via DNA-targeting systems, such as zinc fingers (ZFs), transcription activator-like effectors (TALEs), or clustered regularly interspaced short palindromic repeats (CRISPRs) (5, 8,17,33). Compared to artificial transcription factors (ATFs), which exploit programmable DNA-binding platforms to target transcriptional activators or repressors with no catalytic domain (such as super KRAB Domain, SKD), epigenetic editing has the promise to induce stable and inheritable gene modulation (4,31). In this study, we provide proof-of-concept that SPDEF provides a promising target for epigenetic editing to prevent epithelial MUC5AC expression.…”

Section: Introductionmentioning

confidence: 99%

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Targeted epigenetic editing of SPDEF reduces mucus production in lung epithelial cells

Song

Cano-Rodriquez

Winkle

et al. 2017

American Journal of Physiology-Lung Cellular and Molecular Phys

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Airway mucus hypersecretion contributes to the morbidity and mortality in patients with chronic inflammatory lung diseases. Reducing mucus production is crucial for improving patients' quality of life. The transcription factor SAM-pointed domain-containing Ets-like factor () plays a critical role in the regulation of mucus production and, therefore, represents a potential therapeutic target. This study aims to reduce lung epithelial mucus production by targeted silencing using the novel strategy, epigenetic editing. Zinc fingers and CRISPR/dCas platforms were engineered to target repressors (KRAB, DNA methyltransferases, histone methyltransferases) to the promoter. All constructs were able to effectively suppress both mRNA and protein expression, which was accompanied by inhibition of downstream mucus-related genes [anterior gradient 2 (), mucin 5AC ()]. For the histone methyltransferase G9A, and not its mutant or other effectors, the obtained silencing was mitotically stable. These results indicate efficient silencing and downregulation of mucus-related gene expression by epigenetic editing, in human lung epithelial cells. This opens avenues for epigenetic editing as a novel therapeutic strategy to induce long-lasting mucus inhibition.

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Section: Discussionmentioning

confidence: 80%

Section: Plasmids Constructsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Targeted epigenetic editing of SPDEF reduces mucus production in lung epithelial cells

Song

Cano-Rodriquez

Winkle

et al. 2017

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…For example, dCas9-SAM can be used as a means to activate HIV-1 provirus as discussed above (Zhang et al, 2015a; Bialek et al, 2016). dCas9 derivatives can also be used to execute target-specific epigenetic changes such as DNA methylation and histone acetylation (Cano-Rodriguez and Rots, 2016). For example, dCas9 has been fused to TET1 DNA demethylase catalytic domain and used to target the BRCA1 promoter where it caused active DNA demethylation and upregulation of BRCA1 (Choudhury et al, 2016).…”

Section: Recent Applications Of Crisprmentioning

confidence: 99%

CRISPR Editing Technology in Biological and Biomedical Investigation

White

Kaminski

Young

et al. 2017

J of Cellular Biochemistry

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The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to confer provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation. The power of CRISPR lies in its simplicity and ease of use, its flexibility to be targeted to any given nucleotide sequence by the choice of an easily synthesized guide RNA, and its ready ability to continue to undergo technical improvements. Applications for CRISPR are numerous including creation of novel transgenic cell animals for research, high-throughput screening of gene function, potential clinical gene therapy and nongene-editing approaches such as modulating gene activity and fluorescent tagging. In this prospect article, we will describe the salient features of the CRISPR system with an emphasis on important drawbacks and considerations with respect to eliminating off-target events and obtaining efficient CRISPR delivery. We will discuss recent technical developments to the system and we will illustrate some of the most recent applications with an emphasis on approaches to eliminate human viruses including HIV-1, JCV and HSV-1 and prospects for the future.

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“…Structurebased models have informed targeted knockdown of chromatin subunits and the rational design of low molecular weight inhibitor compounds (reviewed in [1] ). DNAbinding domains fused with structural chromatin proteins and histonemodifying enzymes have been used to generate ectopic chromatin conformations at specific loci [2,3] . Until recently, scientists have not yet leveraged PTMbinding peptides from natural effector proteins to "read" the rich biological information encoded in histone marks in living cells.…”

Section: Introductionmentioning

confidence: 99%

Tandem histone-binding domains enhance the activity of a synthetic chromatin effector

Tekel

Vargas

Song

et al. 2017

Preprint

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Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than DNA sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. We have previously reported the development and validation of the "Polycomb based transcription factor" (PcTF), a fusion protein that recognizes histone modifications through a protein protein interaction between its polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at genomic sites. We demonstrated that PcTF activates genes at methyl histone enriched loci in cancer derived cell lines. However, PcTF induces modest activation of a methyl histone associated reporter compared to a DNA binding activator. Therefore, we modified PcTF to enhance its target affinity. Here, we demonstrate the activity of a modified regulator called Pc 2 TF, which has two tandem copies of the H3K27me3 binding PCD at the N terminus. Pc 2 TF shows higher affinity for H3K27me3 in vitro and shows enhanced gene activation in HEK293 cells compared to PcTF.These results provide compelling evidence that the intrinsic histone binding activity of the PCD motif can be used to tune the activity of synthetic histone binding transcriptional regulators.

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Epigenetic Editing: On the Verge of Reprogramming Gene Expression at Will

Cited by 54 publications

References 97 publications

Targeted epigenetic editing of SPDEF reduces mucus production in lung epithelial cells

Targeted epigenetic editing of SPDEF reduces mucus production in lung epithelial cells

CRISPR Editing Technology in Biological and Biomedical Investigation

Tandem histone-binding domains enhance the activity of a synthetic chromatin effector

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