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Related Experiment Video

Updated: May 16, 2025

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots
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Heritable virus-induced germline editing in tomato.

Youngbin Oh1, Ugrappa Nagalakshmi2, Douglas Dahlbeck1

  • 1Innovative Genomics Institute, University of California, Berkeley, California, 94720, USA.

The Plant Journal : for Cell and Molecular Biology
|March 31, 2025
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Summary

Virus-induced genome editing (VIGE) successfully created heritable mutations in tomato. Optimizing promoters and environmental conditions significantly enhanced genome editing efficiency in established Cas9-expressing tomato lines.

Keywords:
YAO promoterheritable plant genome editingtobacco rattle virus‐induced genome editingtomato

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Area of Science:

  • Plant biotechnology
  • Genome editing
  • Molecular biology

Background:

  • Genome editing offers precise genetic modification in plants.
  • Virus-induced genome editing (VIGE) provides a transient method for gene modification.
  • Establishing efficient heritable VIGE in tomato is crucial for crop improvement.

Purpose of the Study:

  • To implement and optimize heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum).
  • To evaluate the efficiency of different promoters (35S, SlRPS5A, SlYAO) for Cas9 expression in VIGE.
  • To investigate the impact of environmental conditions on heritable editing frequencies.

Main Methods:

  • Generated transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under three different promoters.
  • Utilized a tobacco rattle virus (TRV)-based system with guide RNAs (gRNAs) fused to mobile RNA sequences for gene targeting.
  • Targeted Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes for editing.
  • Assessed somatic and heritable editing efficiencies in SpCas9-expressing tomato lines.
  • Optimized environmental conditions, including reduced light intensity, to enhance editing frequencies.

Main Results:

  • Significant somatic editing efficiency was observed in all tested tomato lines targeting SlPDS and SlDMR6 genes.
  • Heritable monoallelic mutations were detected in progenies from SlYAO promoter-driven SpCas9 lines (3% frequency).
  • Optimization of environmental conditions, particularly reduced light intensity, dramatically increased heritable editing frequencies (up to 86% for SlPDS and 100% for SlDMR6).
  • Biallelic mutations were successfully generated under optimized conditions.

Conclusions:

  • The choice of promoter and optimized environmental conditions are critical for enhancing heritable VIGE efficiency in tomato.
  • VIGE offers a promising strategy for generating stable, heritable mutations in tomato without requiring extensive tissue culture or transformation post-establishment of Cas9-expressing lines.
  • This method facilitates efficient mutant generation in established tomato lines, accelerating crop breeding and research.