Faculty Opinions recommendation of Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.

2021 ◽  
Author(s):  
Neil Brockdorff ◽  
Guifeng Wei
Keyword(s):  
Epigenome Editing ◽  
Genome Wide ◽  
Transcriptional Memory

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing

Cell ◽  
2021 ◽  
Author(s):  
James K. Nuñez ◽  
Jin Chen ◽  
Greg C. Pommier ◽  
J. Zachery Cogan ◽  
Joseph M. Replogle ◽  
...  
Keyword(s):  
Epigenome Editing ◽  
Genome Wide ◽  
Transcriptional Memory

scholarly journals CRISPR Tools for Physiology and Cell State Changes: Potential of Transcriptional Engineering and Epigenome Editing

2021 ◽  
Vol 101 (1) ◽  
pp. 177-211
Author(s):  
Christopher T. Breunig ◽  
Anna Köferle ◽  
Andrea M. Neuner ◽  
Maximilian F. Wiesbeck ◽  
Valentin Baumann ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Molecular Mechanisms ◽  
New Technologies ◽  
Epigenome Editing ◽  
Dna Targeting ◽  
Genome Wide ◽  
Transcriptional Engineering ◽  
Genome Wide Data ◽  
Experimental Approaches ◽  
State Changes

Given the large amount of genome-wide data that have been collected during the last decades, a good understanding of how and why cells change during development, homeostasis, and disease might be expected. Unfortunately, the opposite is true; triggers that cause cellular state changes remain elusive, and the underlying molecular mechanisms are poorly understood. Although genes with the potential to influence cell states are known, the historic dependency on methods that manipulate gene expression outside the endogenous chromatin context has prevented us from understanding how cells organize, interpret, and protect cellular programs. Fortunately, recent methodological innovations are now providing options to answer these outstanding questions, by allowing to target and manipulate individual genomic and epigenomic loci. In particular, three experimental approaches are now feasible due to DNA targeting tools, namely, activation and/or repression of master transcription factors in their endogenous chromatin context; targeting transcription factors to endogenous, alternative, or inaccessible sites; and finally, functional manipulation of the chromatin context. In this article, we discuss the molecular basis of DNA targeting tools and review the potential of these new technologies before we summarize how these have already been used for the manipulation of cellular states and hypothesize about future applications.

scholarly journals Genome-wide profiling of an enhancer-associated histone modification reveals the influence of asthma on the epigenome of the airway epithelium

2018 ◽  
Author(s):  
Peter McErlean ◽  
Audrey Kelly ◽  
Jaideep Dhariwal ◽  
Max Kirtland ◽  
Julie Watson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Histone Modification ◽  
Molecular Mechanisms ◽  
Airway Disease ◽  
Epigenome Editing ◽  
Genome Wide ◽  
Genes Encoding ◽  
Chronic Airway Disease ◽  
Dynamic Relationships

AbstractAsthma is a chronic airway disease driven by complex genetic-environmental interactions. The role of epigenetic modifications in bronchial epithelial cells (BECs) in asthma is poorly understood. We undertook genome-wide profiling of the enhancer-associated histone modification H3K27ac in BECs from people with asthma and healthy controls. We identified 49,903 regions exhibiting differential H3K27ac enrichment in asthma, clustered at genes associated with type-2-high asthma (CLCA1) and epithelial processes (EMT). Asthma dramatically influenced the BEC enhancer landscape and we identified asthma-associated Super-Enhancers encompassing genes encoding transcription factors (TP63) and enzymes regulating lipid metabolism (NOX4). We integrated published protein, epigenomic and transcriptomic datasets and identified epithelium-specific transcription factors associated with H3K27ac in asthma (TP73) and dynamic relationships between asthma-associated changes in H3K27ac, DNA methylation, genetic susceptibility and transcriptional profiles. Finally, we used a CRISPR-based approach to recapitulate the H3K27ac-asthma landscape in vitro and provide proof of principal that asthma-associated gene expression (SERPINB2) is driven in part by aberrant histone acetylation, validating the combination of genome-wide and epigenome-editing approaches in deciphering the molecular mechanisms underlying asthma pathogenesis.

scholarly journals Epigenome editing strategies for the functional annotation of CTCF insulators

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Daniel R. Tarjan ◽  
William A. Flavahan ◽  
Bradley E. Bernstein
Keyword(s):  
Dna Methyltransferases ◽  
Gene Interactions ◽  
Transcriptional Repressors ◽  
Loss Mechanism ◽  
Topological Constraint ◽  
Epigenome Editing ◽  
Genome Wide ◽  
Topologically Associated Domains ◽  
Hit And Run ◽  
The Impact

Abstract The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs). Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing. Finally, we apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.

scholarly journals Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker

2020 ◽  
Author(s):  
Georgina E.T. Blake ◽  
Xiaohui Zhao ◽  
Hong wa Yung ◽  
Graham J. Burton ◽  
Anne C. Ferguson-Smith ◽  
...  
Keyword(s):  
Congenital Malformations ◽  
Epigenetic Inheritance ◽  
Folate Metabolism ◽  
Transgenerational Epigenetic Inheritance ◽  
Hypomorphic Mutation ◽  
Genome Wide ◽  
A Genome ◽  
Epigenetic Instability ◽  
Transcriptional Memory ◽  
Methylation Defects

ABSTRACTThe mechanism behind transgenerational epigenetic inheritance (TEI) is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrrgt hypomorphic mutation results in TEI of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. In this work, we use a genome-wide approach to reveal genetic stability in the Mtrrgt model and epigenome-wide differential DNA methylation in the germline of Mtrr+/gt maternal grandfathers. While epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional memory of germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos at least until the F3 generation in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.

scholarly journals Transcription factor regulation and chromosome dynamics during pseudohyphal growth

2014 ◽  
Vol 25 (17) ◽  
pp. 2669-2676 ◽  
Author(s):  
David Mayhew ◽  
Robi D. Mitra
Keyword(s):  
Dna Binding ◽  
Dna Looping ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Binding Motif ◽  
Pseudohyphal Growth ◽  
Genome Wide ◽  
Transcription Factor Regulation ◽  
Transcriptional Memory

Pseudohyphal growth is a developmental pathway seen in some strains of yeast in which cells form multicellular filaments in response to environmental stresses. We used multiplexed transposon “Calling Cards” to record the genome-wide binding patterns of 28 transcription factors (TFs) in nitrogen-starved yeast. We identified TF targets relevant for pseudohyphal growth, producing a detailed map of its regulatory network. Using tools from graph theory, we identified 14 TFs that lie at the center of this network, including Flo8, Mss11, and Mfg1, which bind as a complex. Surprisingly, the DNA-binding preferences for these key TFs were unknown. Using Calling Card data, we predicted the in vivo DNA-binding motif for the Flo8-Mss11-Mfg1 complex and validated it using a reporter assay. We found that this complex binds several important targets, including FLO11, at both their promoter and termination sequences. We demonstrated that this binding pattern is the result of DNA looping, which regulates the transcription of these targets and is stabilized by an interaction with the nuclear pore complex. This looping provides yeast cells with a transcriptional memory, enabling them more rapidly to execute the filamentous growth program when nitrogen starved if they had been previously exposed to this condition.

scholarly journals Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects

2020 ◽  
Vol 21 (2) ◽  
pp. 502 ◽  
Author(s):  
Daniel Hofacker ◽  
Julian Broche ◽  
Laura Laistner ◽  
Sabrina Adam ◽  
Pavel Bashtrykov ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Efficiency ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Single Chain ◽  
Genomic Locus ◽  
Epigenome Editing ◽  
Highly Active ◽  
Genome Wide ◽  
Potential Applications

Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the catalytically deactivated Cas9 protein (dCas9)-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L single-chain effector domain, allowing for a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A-DNMT3L variants containing mutations in the DNMT3A part, which reduced their endogenous DNA binding. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation, most pronounced for the R887E mutant. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to future applications of this approach.

scholarly journals Direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping (CasKAS)

2021 ◽  
Author(s):  
Georgi K Marinov ◽  
Samuel H. Kim ◽  
S. Tansu Bagdatli ◽  
Alexandro E. Trevino ◽  
Josh Tycko ◽  
...  
Keyword(s):  
Dna Structure ◽  
Binding Specificity ◽  
Practical Application ◽  
Enzyme Binding ◽  
Epigenome Editing ◽  
Genome Wide ◽  
Cas9 Protein ◽  
Target Activity

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structure formed upon binding of a sgRNA-loaded Cas9 protein (''CasKAS''). We demonstrate this method in both in vitro and in vivo contexts.

scholarly journals Epigenome editing of the potential enhancers of the schizophrenia risk genes: approaches for optimization of the genetic constructs

2019 ◽  
pp. 103-105
Author(s):  
D. A. Abashkin ◽  
A. O. Kurishev ◽  
S. V. Smirnova ◽  
D. S. Karpov ◽  
V. E. Golimbet
Keyword(s):  
Regulation Of Gene Expression ◽  
Genetic Associations ◽  
Promoter Regions ◽  
Epigenome Editing ◽  
Coding Regions ◽  
Genome Wide ◽  
Increased Risk ◽  
Hereditary Factors ◽  
Active Transcription ◽  
Genetic Constructs

Hereditary factors contribute significantly to the development of schizophrenia. However, despite many years of research, the genetic architecture and mechanisms of the participation of genetic factors in the development of schizophrenia are not well understood. Genome-wide analyzes of genetic associations in various non-coding regions of the genome, including gene enhancers, revealed many loci associated with an increased risk of schizophrenia. In the course of the analysis of the spatial structure of the genome, we revealed the interaction of these enhancers with the promoter regions of genes involved in the metabolism of neurons. To study in more detail the functions of these genes and the participation of enhancers in their regulation, we obtained plasmid and lentiviral constructs of a functionally active transcription repressor based on the CRISPR / SpyCas9 system, as well as the endonuclease system. The use of these constructs in studies of the functions of enhancers and genes associated with the metabolism and regulation of gene expression in neurons is discussed.

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