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Chromatin-Assisted Targeting Enables Precise DNA Methylation Editing in Plants

Yan HeiD, Ming Wang, Tyler J. BuckleyiD, Brandon A. BooneiD, Erika Li, Janice Yerin Shin, Nicholas Alvarado, Bohao Xu, Anh Nguyen, Shuya WangiD, Yuxing ZhouiD, Suhua FengiD, Steven E. JacobseniD

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Abstract

Precise installation of DNA methylation at selected loci offers a powerful strategy for regulating gene expression without altering DNA sequence, but existing plant epigenome editors are constrained by limited efficiency, locus dependence, and genome-wide off-target methylation. Here, we developed SunTag-MQ1v variants incorporating TRBIP1, which promotes removal of the antagonistic H3K4me3 mark, and CHLAMY, an oligomerizing alpha crystalline domain protein from Chlamydomonas reinhardtii. TRBIP1 enhanced methylation and silencing at the Arabidopsis FWA promoter but caused widespread off-target methylation and severe developmental defects. Adding CHLAMY produced SunTag-CHLAMY-TRBIP1-MQ1v (designated as SunTag-NOVA), which successfully overcame the lethality and widespread off-target effects associated with direct TRBIP1-MQ1v fusions. We demonstrate that CHLAMY drives higher-order oligomerization of the editing complex, which enhances target specificity and mitigates off-target accumulation. SunTag-NOVA robustly installed DNA methylation and repressed transcription at the endogenous FWA, FT and TMM genes with minimal genome-wide off-target consequences. These results show that combining local chromatin modification with controlled effector assembly can improve targeted DNA methylation, and establish SunTag-NOVA as a specific epigenome-editing platform for plants.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (26 Aug 2026) · CC BY 4.0

SunTag-NOVA, a SunTag-MQ1v editor adding TRBIP1 and an oligomerizing CHLAMY domain, methylates and silences FWA, FT and TMM in Arabidopsis without the genome-wide off-target methylation seen with TRBIP1 alone.

Plant epigenome editors are limited by efficiency, locus dependence and genome-wide off-target methylation. The paper reports that dCas9-NtDRMcd causes substantial off-target hypermethylation, and that dCas9-MQ1v has fewer off-target effects but worked only at FWA.

Key results

  1. ShownFusing TRBIP1 to SunTag-MQ1v greatly increased FWA promoter methylation and silencing and gave strong early flowering. It also caused lethality in most plants and extensive off-target methylation.

    WGBS showed off-target CG methylation up to 50% and over 70% methylation of the chloroplast genome, versus SunTag-MQ1v at the same FWA guides; most plants died after bolting without setting seed. · Efficiency enhancement of TRBIP1 in a…; Figure legends

  2. ShownOf five representative ACD proteins fused to SunTag-TRBIP1-MQ1v, only CHLAMY (Chlamydomonas reinhardtii) rescued lethality while keeping strong early flowering. It produced discrete GFP foci, whereas the construct without it was diffuse.

    Candidates were CHLAMY, ZmHSP18, HsHSPB1, HsHSPB4 and HsHSPB8, tested in the fwa rdr6 background; foci were scored by root-tip confocal imaging. The paper reports this qualitatively. · Engineering diverse ACD proteins for the…; Figure legends

  3. ShownSunTag-NOVA (SunTag-CHLAMY-TRBIP1-MQ1v) methylated the FWA promoter with little spread to adjacent regions. Genome-wide and chloroplast off-target methylation were virtually eliminated relative to SunTag-TRBIP1-MQ1v.

    WGBS of T1 plants in fwa; comparison with SunTag-MQ1v and SunTag-TRBIP1-MQ1v genome browser tracks. No percentage is given for the NOVA lines. · The addition of CHLAMY to SunTag-TRBIP1-MQ1v…; Figure legends

  4. ShownSunTag-NOVA also removed H3K4me3 at the FWA promoter. The 1,217 off-target H3K4me3 loss sites seen with dCas9-TRBIP1-MQ1v were largely absent.

    H3K4me3 ChIP-seq in T2 plants compared with the fwa epiallele; the off-target set comes from re-analysing earlier dCas9-TRBIP1-MQ1v data (signalValue > 1.5 relative to fwa). · The addition of CHLAMY to SunTag-TRBIP1-MQ1v…; Figure legends

  5. ShownIn wild-type plants, SunTag-NOVA methylated the FT and TMM promoters and lowered their expression. This produced late flowering (FT) and stomatal clustering (TMM), with genome-wide methylation profiles nearly indistinguishable from wild type.

    RT-qPCR in independent T1 lines (n = 3, P < 0.0001, two-tailed t-test); stomatal clusters quantified with n = 10. At FT, methylation peaked about 1.5 kb downstream of the gRNA site. · SunTag-NOVA enables targeted DNA methylation…; Figure legends

Methods, data and reproducibility

The authors added TRBIP1 (an H3K4me3-removal recruiter) and CHLAMY (an alpha-crystallin-domain oligomerizing protein) to a dCas9 SunTag-MQ1v CG methyltransferase system. They tested T1/T2 Arabidopsis lines using McrBC-qPCR, bisulfite sequencing (BS-PCR and WGBS), H3K4me3 ChIP-seq, RT-qPCR and confocal imaging.

  • WGBS of T1 transgenic leaves (SunTag-MQ1v, SunTag-TRBIP1-MQ1v, SunTag-NOVA at FWA, FT, TMM) versus WT and fwa (Fig. 1G comparison) · Arabidopsis thaliana · n = 2 lines each (MQ1v, TRBIP1-MQ1v); NOVA n unstated; ≥24 lines made per construct
  • H3K4me3 ChIP-seq in fwa and SunTag-NOVA_FWA-gRNA T2 plants, plus re-analysis of earlier dCas9-TRBIP1-MQ1v ChIP-seq · Arabidopsis thaliana
  • BS-PCR sequencing of three FWA promoter regions; McrBC-qPCR and RT-qPCR · Arabidopsis thaliana · n = n = 3 (qPCR)
  • Root-tip confocal imaging of SunTag-ACD scFv-GFP foci and stomatal cluster counts · Arabidopsis thaliana · n = n = 10 (stomatal clusters)
Methods & tools
SunTag, dCas9, MQ1v, TRBIP1, CHLAMY, WGBS, H3K4me3 ChIP-seq, McrBC-qPCR, AlphaFold3, ChimeraX v1.10.1, ViewBS v0.1.11, yeast two-hybrid
Reagents & environment
ViewBS v0.1.11 for methylation visualization; ChimeraX v1.10.1 for structure visualization; WGBS analysed with a previously described pipeline (not detailed here).
Reproducibility gaps
Sequencing data are said to be deposited in GEO, but the text marks this as still to be uploaded and gives no accession. No code repository is given.
Limitations (2)
  • At FT, methylation spread beyond the gRNA target site, peaking about 1.5 kb downstream; the authors attribute this to the multivalent SunTag plus CHLAMY assembly, which could reduce locus precision. · noted in this summary
  • The two mechanisms (higher local effector concentration and sequestration of unbound effector) are only proposed in the discussion; the foci imaging, AlphaFold3 and yeast two-hybrid data shown do not test sequestration directly. · noted in this summary

Read this if you work on plant epigenome editing and want a CG methylation editor with fewer genome-wide off-target effects, or a way to curb effector off-targeting by oligomerization.

Citations
1

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