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Nature PlantsPlant BiologyPeer-reviewed journal article

High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB

Ugrappa NagalakshmiiD, Jorge E. RodrigueziD, Thi Nguyen, Rachel F. WeissmaniD, Brittney W. ThorntoniD, Cynthia I. TerraceiD, David F. SavageiD, Savithramma P Dinesh-KumariD

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Abstract

Genome editing has revolutionized plant biology research. However, efficient and straightforward delivery of editing reagents remains a major challenge. Viral delivery systems can address these issues, but CRISPR-Cas nucleases are often too large for viral vectors. Recently, smaller editors like TnpBs have been identified, but wild-type TnpBs are significantly less active than commonly used Cas9 nucleases. Here, we optimized a tobacco rattle virus (TRV)-based system to deliver newly discovered, highly active engineered ISDra2 TnpB variants. Our results demonstrate that the eTnpBc variant delivered via TRV enables effective somatic editing in systemic leaves and achieves up to 90% editing efficiency at target loci, significantly higher than that of wild-type ISDra2 TnpB. Additionally, up to 89% of offspring exhibit a mutant phenotype, with editing efficiencies reaching 100%. The design principles outlined here are expected to accelerate broader adoption of eTnpBc for transformation- and transgene-free genome editing in plants.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (9 Dec 2025) · CC BY 4.0

Tobacco rattle virus delivery of engineered ISDra2 variant eTnpBc and its guide RNA as a single transcript achieves up to 90% somatic and 100% heritable editing in Nicotiana benthamiana.

Plant viral vectors can bypass labor-intensive tissue culture transformation for transgene-free genome editing, but their limited cargo capacity excludes Cas9 and wild-type compact TnpB nucleases have low editing activity. Delivering deep-mutational-scanning-engineered eTnpBc with its omega-RNA (reRNA) in a single tobacco rattle virus (TRV) vector enables high-efficiency somatic and germline editing.

Key results

  1. ShownEncoding the engineered ISDra2 variant eTnpBc and its guide RNA (reRNA) as a single transcript in TRV2 induces strong systemic somatic editing at the Phytoene desaturase (PDS) locus in Nicotiana benthamiana, unlike two-vector delivery or wild-type TnpB.

    Delivering TnpB and reRNA4^PDS on separate TRV2 vectors yielded <0.7% systemic leaf indels; as a single transcript under pPEBV, eTnpBc caused systemic photobleaching in 88% of plants (14/16) with systemic leaf indel frequencies of 71+-9% (reRNA4^PDS) and 62+-17% (^HHreRNA4^PDS), compared to 26+-4% and 20+-4% for eTnpBe and 3+-1% and 12+-4% for wild-type TnpB (n=4 biological replicates). · Main; Figure legends

  2. ShownHuman-yeast co-optimized eTnpBc outperforms plant codon-optimized eTnpBc for systemic editing and achieves 90% systemic somatic editing at the magnesium chelatase (ChlH) homeologs.

    While N. benthamiana codon-optimized NbCoeTnpBc achieved 55+-8% and 56+-6% editing in infiltrated leaves, it caused only mild systemic photobleaching; human-yeast co-optimized eTnpBc targeting two ChlH homeologs (ChlH1 and ChlH2) produced yellow systemic leaves with 90+-12% systemic indel frequency (n=6 biological replicates). · Main; Figure legends

  3. ShownTRV-delivered eTnpBc produces high-frequency, virus-free, heritable tetra-allelic mutations in M1 progeny at both PDS and ChlH loci without detectable off-target indels.

    Seedlings from photobleached or yellow pods were 89+-9% (reRNA4^PDS), 89+-8% (^HHreRNA4^PDS), and 100% (reRNA^ChlH) mutant (52-58% from pooled top-third pods vs 0% for WT TnpB or eTnpBe), with 88-96% PDS and 99-100% ChlH indel frequencies, no RT-PCR-detectable TRV in progeny, and 0 indels across 7 PDS and 6 ChlH Cas-OFFinder sites (n=5 seedlings each). · Main; RT-PCR; Off-target analysis; Figure legends

  4. ShownEngineered ISYmu1 TnpB variants (H4Y/V305R and L167G/V305R) delivered via TRV2 also induce systemic PDS photobleaching in all infiltrated N. benthamiana plants, whereas wild-type ISYmu1 does not.

    Wild-type ISYmu1 (Arabidopsis, N. tabacum, or human/yeast codon-optimized) produced 24+-3% to 33+-2% editing in infiltrated leaves but failed to induce systemic photobleaching, whereas both engineered ISYmu1^(H4Y/V305R) and ISYmu1^(L167G/V305R) induced systemic photobleaching across all infiltrated plants. · Main

Methods, data and reproducibility

Cloned wild-type and engineered ISDra2 TnpB (eTnpBc, eTnpBe) and ISYmu1 variants fused to FDnls or 2xSV40nls, together with ribozyme-flanked reRNAs and tRNA^Ile, into TRV2 downstream of the pPEBV promoter. Agroinfiltrated 2.5-3-week-old Nicotiana benthamiana leaves alongside TRV1 and quantified somatic and M1 progeny editing at PDS and ChlH homeologs using Amplicon-EZ sequencing and CRISPResso2.

  • Infiltrated leaves, systemic leaves, and M1 progeny seedlings of allotetraploid Nicotiana benthamiana edited at PDS (Nbe06g25970, Nbe05g35010) and ChlH (Nbe07g00760, Nbe08g21240) loci · plant (other) (Nicotiana benthamiana) · n = n=4-6 leaf replicates; 6-8 M1 seedlings/parent; 5 seedlings/locus off-target
Methods & tools
CRISPResso2, Cas-OFFinder, NEBuilder HiFi DNA Assembly
Reproducibility gaps
Amplicon-EZ sequencing files are stated to be accessible on the NCBI Sequence Read Archive only 'at the time of publication' without an accession number provided (S11).
Limitations (2)
  • The eTnpBc viral system generates indels rather than precise base or prime edits, and testing was conducted on genes with visual loss-of-function phenotypes (PDS and ChlH) in Nicotiana benthamiana rather than crop species (S2). · stated by the authors
  • Plant codon-optimized versions of eTnpBc failed to induce strong systemic photobleaching despite similar viral and TnpB transcript levels by qPCR, potentially due to differences in protein stability or loss of viral insert sequences during systemic spread (S2). · stated by the authors

Read this if you develop viral delivery vectors, compact RNA-guided TnpB nucleases, or tissue-culture-free heritable genome editing methods in plants.

Citations
22
FWCI
45.15
Field percentile
top 1%

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