Efficient recovery of transgene-free edited tobacco using the transgene self-elimination system via de novo regeneration
- 1Tobacco Research Institute, Chinese Academy of Agricultural Sciences
Abstract
Elimination of integrated editing constructs remains a major challenge for generating transgene-free progeny in plant genome editing based on Agrobacterium-mediated regeneration. Here, we adapted the Transgene Killer CRISPR (TKC) concept to tobacco and established the transgene self-elimination system, in which CRISPR/Cas9 is coupled with developmentally regulated BARNASE cassettes. Three pU3TR-derived vectors carrying DLL-, OsRP3- or PHAS-driven Barnase regenerated normally after leaf-disc transformation. The DLL construct pEB138 showed the highest T editing frequency, with 56 of 69 independent T plants (81.16%) carrying mutations at one or both NtDFR target loci. Under a stringent lineage-level screen in which up to 48 T progeny were tested per T line, 36 of 43 pEB138 lineages (83.72%) yielded uniformly marker-negative progeny for the three assayed T-DNA markers, compared with 0 of 7 lineages transformed with the parental pU3TR vector. The platform also supported recovery of dfr mutants with altered flower pigmentation and NtA622-edited lines with reduced nicotine accumulation. Moreover, introduction of DLL-iBarnase into the independent pDC45 editing backbone did not impair shoot regeneration, and representative NtBBL progeny showed markedly reduced nicotine content together with loss of the selectable cassette. These results demonstrate that BARNASE-assisted transgene elimination can be integrated with Agrobacterium-mediated de novo regeneration in a dicot, enabling one-step recovery of transgene-free edited tobacco progeny.
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BibTeX
@misc{zhang2026efficient,
title = {{Efficient recovery of transgene-free edited tobacco using the transgene self-elimination system via de novo regeneration}},
author = {J. Zhang and W. Li and J. Lv and X. Liu and L. Wang and Y. Qian and Y. Liu and F. Qi and H. Xue and X. Liu and L. Deng and F. Wang and J. Xu and K. Xue and X. Liu and Q. Wu and H. Xiang and C. Dai},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.24.761527},
url = {https://www.biorxiv.org/content/10.64898/2026.10.24.761527v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Efficient recovery of transgene-free edited tobacco using the transgene self-elimination system via de novo regeneration
AU - J. Zhang
AU - W. Li
AU - J. Lv
AU - X. Liu
AU - L. Wang
AU - Y. Qian
AU - Y. Liu
AU - F. Qi
AU - H. Xue
AU - X. Liu
AU - L. Deng
AU - F. Wang
AU - J. Xu
AU - K. Xue
AU - X. Liu
AU - Q. Wu
AU - H. Xiang
AU - C. Dai
PY - 2026
DA - 2026/10/24
PB - bioRxiv
DO - 10.64898/2026.10.24.761527
UR - https://www.biorxiv.org/content/10.64898/2026.10.24.761527v1
AB - Elimination of integrated editing constructs remains a major challenge for generating transgene-free progeny in plant genome editing based on Agrobacterium-mediated regeneration. Here, we adapted the Transgene Killer CRISPR (TKC) concept to tobacco and established the transgene self-elimination system, in which CRISPR/Cas9 is coupled with developmentally regulated BARNASE cassettes. Three pU3TR-derived vectors carrying DLL-, OsRP3- or PHAS-driven Barnase regenerated normally after leaf-disc transformation. The DLL construct pEB138 showed the highest T editing frequency, with 56 of 69 independent T plants (81.16%) carrying mutations at one or both NtDFR target loci. Under a stringent lineage-level screen in which up to 48 T progeny were tested per T line, 36 of 43 pEB138 lineages (83.72%) yielded uniformly marker-negative progeny for the three assayed T-DNA markers, compared with 0 of 7 lineages transformed with the parental pU3TR vector. The platform also supported recovery of dfr mutants with altered flower pigmentation and NtA622-edited lines with reduced nicotine accumulation. Moreover, introduction of DLL-iBarnase into the independent pDC45 editing backbone did not impair shoot regeneration, and representative NtBBL progeny showed markedly reduced nicotine content together with loss of the selectable cassette. These results demonstrate that BARNASE-assisted transgene elimination can be integrated with Agrobacterium-mediated de novo regeneration in a dicot, enabling one-step recovery of transgene-free edited tobacco progeny.
ER -