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Review
. 2023 Dec 21;4(1):40-57.
doi: 10.1021/jacsau.3c00671. eCollection 2024 Jan 22.

Reshaping the Landscape of the Genome: Toolkits for Precise DNA Methylation Manipulation and Beyond

Chenyou Zhu  1 , Ziyang Hao  2 , Dongsheng Liu  1
Affiliations
Review

Reshaping the Landscape of the Genome: Toolkits for Precise DNA Methylation Manipulation and Beyond

Chenyou Zhu et al. JACS Au. .

Abstract

DNA methylation plays a pivotal role in various biological processes and is highly related to multiple diseases. The exact functions of DNA methylation are still puzzling due to its uneven distribution, dynamic conversion, and complex interactions with other substances. Current methods such as chemical- and enzyme-based sequencing techniques have enabled us to pinpoint DNA methylation at single-base resolution, which necessitated the manipulation of DNA methylation at comparable resolution to precisely illustrate the correlations and causal relationships between the functions of DNA methylation and its spatiotemporal patterns. Here a perspective on the past, recent process, and future of precise DNA methylation tools is provided. Specifically, genome-wide and site-specific manipulation of DNA methylation methods is discussed, with an emphasis on their principles, limitations, applications, and future developmental directions.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Representative DNMTi and effect mechanism. (A) Chemical structures of cytosine-like DNMTi. (B) Scheme of the mechanism of 5-Aza in inhibiting the activity of DNMT. In a typical reaction of DNMT with normal cytosine, the cysteine residue of DNMT will mediate a nucleophilic attack on the cytosine ring with the facilitation of another glutamic acid residue of DNMT. Then the methyl group of S-adenosylmethionine (SAM) (the typical methyl group donor) will be transferred on the C5 of the cytosine ring with the help of the base provided by DNMT. In the methylation process of cytosine, DNMT will be released from the covalent complex and conduct enzymatic reaction on other cytosines. However, with the addition of 5-Aza and incorporation of it into the genome, 5-Aza will irreversibly bind with DNMT, which will inhibit the activity of DNMT and send it to be digested.
Figure 2
Figure 2
A typical workflow of using genetic perturbation to study the functions of 5mC.
Figure 3
Figure 3
Scheme of the construction of fusion proteins for targeted DNA methylation manipulation. Shown is an example of targeted methylation. In accordance with the types of programmable DNBPs, fusion proteins can be categorized into those based on ZF, TALE, and CRISPR/Cas9 fusion proteins.
Figure 4
Figure 4
Scheme of dCas9-based fusion proteins for targeted DNA methylation. (A) The common construction of dCas9-based fusion proteins, which involves linking the dCas9 with the catalytic domain of de novo methyltransferase like DNMT3A to enable targeted DNA methylation., Reproduced with permission from ref (104). Copyright 2016 Oxford University Press. (B) Scheme of multiplexed sgRNA-targeting for long-range targeted methylation. Pooled sgRNAs are used for multilocus targeting and enabling long-range targeted methylation. Reproduced with permission from ref (104). Copyright 2016 Oxford University Press. (C) Scheme of targeted methylation using the dCas-SunTag system. To enable long-range methylation with only a single sgRNA, dCas9 is fused with a SunTag array, which is an amplifier based on the array of multiple antibody epitopes. DNMT3A is fused with scFv, which can recognize the SunTag unit, and the in situ enrichment of multiple copies of DNMT3A-scFv to broaden the effective region., Reproduced with permission from ref (108). Copyright 2017 Springer Nature. (D) Scheme of reduction of off-target effect by split-methyltransferase. dCas9 is fused with C-terminus of methyltransferase M.SssI (M.SssI[273–386]) and only the simultaneous enrichment of dCas9-M.SssI[273–386] and M.SssI[1–272] at targeted sites can reactivate M.SssI, which will theoretically increase the specificity of the system. Reproduced with permission from ref (110). Copyright 2017 Springer Nature.
Figure 5
Figure 5
Scheme of the generation of off-target effects of the fusion protein. Shown is the taking targeted methylation by dCas9-based fusion proteins as an example. (A) Scheme of DNBP-based off-target effect. In the dCas9 system, the mismatch between sgRNA and target DNA could be tolerated, which will recruit dCas9-DNMT fusion protein to undesired sites, which will result in the off-target effect. (B) Scheme of effector protein-based off-target effect. The effector proteins such as DNMTs could exhibit enzymatic activity at undesired sites which generates off-target efficiency. (C) Scheme of off-target effect generated from genome high-order structures. 3D genome structures could bring distantly located genomic regions into close proximity, and the flexibility of the linker will enable the flipping of effector proteins between on-target sites and off-target sites, which generates the off-target effect.
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