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Background <p>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.</p> Results <p>To address these limitations, we used <i>Medicago truncatula</i> 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 <i>MtABCG46</i> 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 <i>Agrobacterium tumefaciens</i>–mediated transformation yielded heritable mutations, and transgene-free knockout lines were recovered in subsequent generations.</p> Conclusions <p>This study establishes a robust hairy root-based gRNA validation platform for <i>M. truncatula</i>, generates stable and heritable <i>mtabcg46</i> knockout lines, and provides a scalable framework for functional characterization of ABCG transporters in specialized metabolism and plant defense.</p>

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Generation of transgene-free MtABCG46 mutants in Medicago truncatula using CRISPR/Cas9

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月4日

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  • Praveen Awasthi,
  • Aleksandra Pawela,
  • Krishnapriya Anirudhan,
  • Michał Jasiński

摘要

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.