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Efficient base editing in tomato using a highly expressed transient system.

Shaoze Yuan1, Shunsuke Kawasaki1, Islam M Y Abdellatif1

  • 1Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 305-8572, Japan.

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|February 7, 2021
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Summary

Base editing in tomatoes was successfully achieved using a transient expression system, yielding homozygous mutants efficiently. This method also resulted in nCas9-free next-generation plants, overcoming key challenges in tomato genome editing.

Keywords:
Solanum lycopersicumTarget-AIDTomatoTransient expressionTsukuba system

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Area of Science:

  • Plant Science
  • Genetics
  • Biotechnology

Background:

  • Tomatoes (Solanum lycopersicum) are economically significant and serve as a model for fruit ripening studies.
  • Genome editing technologies like CRISPR/Cas9 are valuable for functional genetic research in plants.
  • Existing genome editing methods in tomatoes face challenges such as low transformation efficiency and the need to remove foreign DNA.

Purpose of the Study:

  • To improve base editing technology in tomatoes by utilizing a high-level transient expression system.
  • To achieve efficient generation of homozygous base-edited tomato mutants.
  • To obtain nCas9-free next-generation plants after base editing.

Main Methods:

  • Established a high-level transient expression system in tomato using geminiviral replication and a double terminator.
  • Introduced nCas9-CDA and sgRNA-SlHWS into the pBYR2HS vector for transient expression.
  • Employed agroinfiltration for transient expression of base editing components in tomato tissues.

Main Results:

  • Achieved base editing in tomatoes via transient expression.
  • Obtained homozygous mutants with an efficiency of approximately 18% in the next generation.
  • Generated nCas9-free next-generation plants with 71% efficiency, with all homozygous base-edited plants being nCas9-free.

Conclusions:

  • The high-level transient expression system is effective for base editing in tomatoes.
  • This system facilitates the generation of homozygous base-edited plants that are free of foreign DNA.
  • Transient expression offers a promising approach to overcome limitations in tomato genome editing.