DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine

Devesa-Guerra, Iván; Morales‐Ruíz, Teresa; Perez-Roldan, Juan; Parrilla-Doblas, Jara Teresa; Dorado-León, Macarena; García-Ortiz, María Victoria; Ariza, Rafael R.; Roldán-Arjona, Teresa

doi:10.1016/j.jmb.2020.02.007

Cited by 42 publications

(27 citation statements)

References 80 publications

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“…Recently (Taghbalout et al, 2016) used Casilio-ME for RNA-guided editing of 5-mC by targeting TET1 to specific genomic sites, and co-delivery of TET1 and other protein factors, to activate methylation-silenced genes. Similarly, Devesa-Guerra et al (2020) fused the catalytic domain of ROS1 5-mC DNA glycosylase with dCas9 and reported that dCas9-ROS1 (but not the dCas9-TET1) can reactivate methylation-silenced genes by active demethylation.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Dynamics of DNA Methylation and Its Functions in Plant Growth and Development

2021

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Epigenetic modifications in DNA bases and histone proteins play important roles in the regulation of gene expression and genome stability. Chemical modification of DNA base (e.g., addition of a methyl group at the fifth carbon of cytosine residue) switches on/off the gene expression during developmental process and environmental stresses. The dynamics of DNA base methylation depends mainly on the activities of the writer/eraser guided by non-coding RNA (ncRNA) and regulated by the developmental/environmental cues. De novo DNA methylation and active demethylation activities control the methylation level and regulate the gene expression. Identification of ncRNA involved in de novo DNA methylation, increased DNA methylation proteins guiding DNA demethylase, and methylation monitoring sequence that helps maintaining a balance between DNA methylation and demethylation is the recent developments that may resolve some of the enigmas. Such discoveries provide a better understanding of the dynamics/functions of DNA base methylation and epigenetic regulation of growth, development, and stress tolerance in crop plants. Identification of epigenetic pathways in animals, their existence/orthologs in plants, and functional validation might improve future strategies for epigenome editing toward climate-resilient, sustainable agriculture in this era of global climate change. The present review discusses the dynamics of DNA methylation (cytosine/adenine) in plants, its functions in regulating gene expression under abiotic/biotic stresses, developmental processes, and genome stability.

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Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Dynamics of DNA Methylation and Its Functions in Plant Growth and Development

2021

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“…Moreover, TET proteins are also able to oxidize thymine to 5-hydroxymethyluracil, thereby introducing another confounding epigenetic mark that produces a unique spectrum of modifications on chromatin structure and transcription factor activity [96]. A recent candidate for the improvement of such a strategy is the fusion of dCas9 to the Arabidopsis ROS1 glycosylase that directly removes 5-methylcytosine by direct base excision repair, foregoing the intermediate oxidized derivatives with epigenetic potential [97]; yet the issue of the overexpression of an enzyme with a capacity for unwanted and non-targeted effects is not solved by this approach.…”

Section: Discussionmentioning

confidence: 99%

Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

Sapozhnikov

Szyf

2020

Preprint

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Although associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are confounded and fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA methylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.

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“…Interestingly, ROS1 and DME can also work in mammalian systems inducing genome‐wide methylation changes and targeted demethylation (Mok et al, 2017; Parrilla‐Doblas et al, 2017; Morales‐Ruiz et al, 2018). Fusion of the ROS1 catalytic domain to dCas9 successfully reactivated a methylation‐silenced luciferase reporter gene and induced partial demethylation in a replication‐independent manner, although the system has not yet been applied to plants (Devesa‐Guerra et al, 2020).…”

Section: Gene Transcription Control and Epigenome Editingmentioning

confidence: 99%

Genome editing for plant research and crop improvement

Zhan¹,

Zhu

et al. 2021

JIPB

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The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control.

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DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine

Cited by 42 publications

References 80 publications

Dynamics of DNA Methylation and Its Functions in Plant Growth and Development

Dynamics of DNA Methylation and Its Functions in Plant Growth and Development

Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

Genome editing for plant research and crop improvement

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