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Molecular CellBiochemistryPeer-reviewed journal article

Stepwise DNA unwinding gates TnpB genome-editing activity

Zehan Zhou, Iren Saffarian-Deemyad, Honglue ShiiD, Trevor Weiss, Muhammad Moez ur-Rehman, Kamakshi VohraiD, Petr SkopintseviD, Peter H. YooniD, Marena I. TrinidadiD, Conner J. LangebergiD, Maris KamaluiD, Jasmine Amerasekera, Yuxing ZhouiD, Erin E. DohertyiD, Kevin Aris, Noor Al-Sayyad, Brittney W. ThorntoniD, Rachel F. WeissmaniD, Kevin M. WaskoiD, Isabel Esain-GarciaiD, Evan C. DeTurkiD, David F. SavageiD, Steven E. JacobseniD, Zev D. BryantiD, Jennifer A. DoudnaiD

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Abstract

TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (18 Jul 2026) · CC BY 4.0

Ymu1 TnpB unwinds DNA via a long-lived partially unwound intermediate before a rarely stable open state; the WFR triple variant stabilizes both, boosting plant editing.

Natural TnpBs edit plant genomes poorly, and the cause of this low activity was unclear even though mutational scans had found activity-enhancing substitutions. This work places the bottleneck in stepwise DNA unwinding and shows that point mutations stabilizing unwound states relieve it.

Key results

  1. ShownThe triple variant Ymu1-WFR (H4W-L304F-V305R) raises editing in Arabidopsis plants well above wild type (WT) Ymu1, which stayed below 10% at all fourteen protoplast loci tested.

    T1 plants at PDS3 g2: ~10-fold (H4W) and ~17-fold (Ymu1-WFR) vs WT; up to 56-fold across seven additional targets (Ymu1-WFR vs WT); WT Ymu1 below 10% editing at all fourteen protoplast loci. · Reconstituted Ymu1 TnpB for mechanistic studies; Ymu1 TnpB mutagenesis identifies determinants of…

  2. ShownThe enhanced variants cleave purified DNA substrates much faster, and the Ymu1-WFR advantage persists with competitor DNA and on a second target.

    kobs from n = 3 reactions (100 nM RNP, 10 nM dsDNA): Ymu1-WFR ~28-fold faster than WT on Target 1; H4W and V305R ~3-fold; L304F not significantly faster; 58-fold with 30-fold mass excess salmon sperm DNA; >5-fold on Target 2. · Ymu1 TnpB variants accelerate DNA cleavage in…; Oligonucleotide cleavage assay

  3. ShownSingle-molecule tracking shows a three-state pathway (closed, intermediate, open). H4W mainly stabilizes the intermediate, while WFR stabilizes both the intermediate and open states.

    All variants show closed, ~7 bp intermediate and ~14 bp open states (100 nM RNP, torsionally relaxed tether). WT sits mostly closed with rare open visits; H4W suppresses I-to-C collapse; WFR suppresses both kI→C and kO→I (Table S3). · AuRBT revealed step-specific enhancement of DNA…

  4. ShownNegative supercoiling compensates for WT's unwinding defect, which largely erases the WT versus WFR cleavage gap on supercoiled plasmid.

    At about -5 Twist (~1% negative supercoiling) the WT intermediate and open states are stabilized. Linearized plasmid: kobs 0.68 (WFR) vs 0.029 min^-1 (WT), 23-fold. Supercoiled: 5.4 vs 3.4 min^-1, 1.6-fold. · Negative supercoiling stabilizes unwound states…

  5. SuggestedYmu1-WFR keeps strong mismatch discrimination in vitro, and no off-target edits were detected in three edited Arabidopsis progeny lines.

    Mismatches at positions 4, 7 and 10 cut WFR kobs >100-fold (0.007-0.011 min^-1). Whole-genome sequencing at 2,406-5,605× found no variants at 82 (CHLI1 g4) or 48 (PDS3 g12) predicted sites; 54 unique variants, none at predicted sites. · Ymu1-WFR shows reduced cleavage on mismatched…

Methods, data and reproducibility

Bulk cleavage kinetics, gold rotor-bead tracking (AuRBT) of catalytically dead RNPs on relaxed and supercoiled DNA with change-point state scoring, and Arabidopsis editing assays, plus structure-guided mutagenesis of 54 variants.

  • Ymu1 editing across endogenous loci in Arabidopsis protoplasts (amplicon sequencing) · Arabidopsis thaliana · n = 14 loci; n = 3 biological replicates · PRJNA1432103
  • Protoplast mutagenesis screen of single and combinatorial Ymu1 variants at PDS3 g2 · Arabidopsis thaliana · n = 54 variants; n = 3 or 6 replicates
  • T1 transgenic plant editing by WT, H4W and Ymu1-WFR (PDS3 g2 plus seven additional targets) · Arabidopsis thaliana · PRJNA1432103
  • In vitro cleavage of 60-bp dsDNA, oligo and 2.2 kb plasmid substrates by Ymu1 variants · Youngiibacter multivorans TnpB (E. coli expressed) · n = n = 3 independent reactions
  • AuRBT single-molecule tracking of dYmu1 variants on ~5-kb DNA tethers · n = number of tethers per Table S4 · 10.25740/qg757fx8501
  • Whole-genome off-target sequencing of transgene-free Ymu1-WFR progeny, with WT Col-0 controls · Arabidopsis thaliana · n = 3 progeny lines; 2 WT controls · PRJNA1482598
  • Bacterial TAM depletion assay, WT vs Ymu1-WFR · Escherichia coli · PRJNA1451272
  • Small RNA-seq and SHAPE-MaP of reRNA within purified RNPs · GSE327273
Methods & tools
AuRBT (gold rotor-bead tracking), Steppi change-point analysis, AlphaFold3, GREMLIN, CrispRvariants v.1.14.0, Cas-OFFinder v3.0.0, BWA-MEM v0.7.17, GATK v4.2.0.0, Strelka2 v2.9.2, ShapeMapper2, PSI-BLAST, GraphPad Prism
Software & environment
TAM analysis in Python 3.8 (Biopython v1.85, pandas v1.4.0, NumPy v1.22.1); MATLAB R2019a; BWA-MEM v0.7.17; GATK v4.2.0.0; Strelka2 v2.9.2; Cas-OFFinder v3.0.0.
Reproducibility gaps
Plasmids will be deposited to Addgene only upon publication; additional reanalysis information is available from the lead contact on request.
Limitations (6)
  • Cleavage assays used purified short oligonucleotide or plasmid substrates, which do not capture target search in cells, including chromatinized genomic DNA. · stated by the authors
  • Genome-editing and off-target experiments were done only in Arabidopsis; extension of enhanced activity and specificity to mammalian or other cells is untested. · stated by the authors
  • The model is built from kinetic and thermodynamic data alone; no high-resolution structure, such as cryo-EM of Ymu1-WFR bound to target DNA, confirms how the mutations act. · stated by the authors
  • The single-molecule model comes from one ortholog (Ymu1) and one target sequence; other targets, mismatches, supercoiling conditions and variants remain untested. · stated by the authors
  • The WT Ymu1 comparator for T1 plant editing at PDS3 g2 was previously published data replotted, not a concurrent control. · noted in this summary
  • The off-target conclusion rests on three progeny lines from one parent plant and two guides, so it supports only absence of detected edits under those conditions. · noted in this summary

Read this if you engineer compact RNA-guided nucleases and want a single-molecule kinetic framework linking R-loop unwinding states to in vitro cleavage and plant editing.

Citations
1
FWCI
2.05
Field percentile
top 10%

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