Generation of transgene-free MtABCG46 mutants in Medicago truncatula using CRISPR/Cas9.
4 affiliations
- 1Department of Plant Molecular Physiology, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, Poznań, 61- 704, Poland.
- 2Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN, 55108, USA.
- 3Department of Plant Molecular Physiology, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, Poznań, 61- 704, Poland. [email protected].
- 4Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Dojazd 11, Poznań, 60-632, Poland. [email protected].
Abstract
Background
ATP-binding cassette (ABC) transporters form one of the largest and most functionally diverse families of membrane proteins in plants, with members of the ABCG subfamily playing central roles in the transport of specialized metabolites and stress adaptation. In legumes functional analyses of these transporters have been limited by functional redundancy, accessibility of T-DNA inserted mutant lines, and the constraints of RNA interference-based approaches.Results
To address these limitations, we used Medicago truncatula ABCG46 transporter and established hairy root-based CRISPR/Cas9 genome-editing platform that enables rapid empirical validation of single guide RNAs prior to stable transformation. Two guide RNAs targeting distinct exons of MtABCG46 were evaluated in 70 independent hairy root lines, revealing strong editing activity for one guide, while the second proved non-functional despite favourable in silico predictions. Sequence analysis identified a range of insertions and deletions, including a homozygous biallelic mutant carrying frame-shifting deletions predicted to abolish transporter function. Using the validated guide RNA, stable Agrobacterium tumefaciens-mediated transformation yielded heritable mutations, and transgene-free knockout lines were recovered in subsequent generations.Conclusions
This study establishes a robust hairy root-based gRNA validation platform for M. truncatula, generates stable and heritable mtabcg46 knockout lines, and provides a scalable framework for functional characterization of ABCG transporters in specialized metabolism and plant defense.
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BibTeX
@article{awasthi2026generation,
title = {{Generation of transgene-free MtABCG46 mutants in Medicago truncatula using CRISPR/Cas9.}},
author = {Praveen Awasthi and Aleksandra Pawela and Krishnapriya Anirudhan and Michał Jasiński},
journal = {Plant methods},
year = {2026},
month = aug,
doi = {10.1186/s13007-026-01582-x},
url = {https://europepmc.org/article/MED/42972389},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Generation of transgene-free MtABCG46 mutants in Medicago truncatula using CRISPR/Cas9.
AU - Praveen Awasthi
AU - Aleksandra Pawela
AU - Krishnapriya Anirudhan
AU - Michał Jasiński
PY - 2026
DA - 2026/08/04
JO - Plant methods
DO - 10.1186/s13007-026-01582-x
UR - https://europepmc.org/article/MED/42972389
AB - Background ATP-binding cassette (ABC) transporters form one of the largest and most functionally diverse families of membrane proteins in plants, with members of the ABCG subfamily playing central roles in the transport of specialized metabolites and stress adaptation. In legumes functional analyses of these transporters have been limited by functional redundancy, accessibility of T-DNA inserted mutant lines, and the constraints of RNA interference-based approaches.Results To address these limitations, we used Medicago truncatula ABCG46 transporter and established hairy root-based CRISPR/Cas9 genome-editing platform that enables rapid empirical validation of single guide RNAs prior to stable transformation. Two guide RNAs targeting distinct exons of MtABCG46 were evaluated in 70 independent hairy root lines, revealing strong editing activity for one guide, while the second proved non-functional despite favourable in silico predictions. Sequence analysis identified a range of insertions and deletions, including a homozygous biallelic mutant carrying frame-shifting deletions predicted to abolish transporter function. Using the validated guide RNA, stable Agrobacterium tumefaciens-mediated transformation yielded heritable mutations, and transgene-free knockout lines were recovered in subsequent generations.Conclusions This study establishes a robust hairy root-based gRNA validation platform for M. truncatula, generates stable and heritable mtabcg46 knockout lines, and provides a scalable framework for functional characterization of ABCG transporters in specialized metabolism and plant defense.
SN - 1746-4811
ER -