Efficient base editing in tomato using a highly expressed transient system

Abstract

Key message

Base editing in tomatoes was achieved by transient expression.

Abstract

The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.

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Abbreviations

AID:

Activation-induced cytidine deaminase

ALS:

Acetolactate synthase

BeYDV:

Bean yellow dwarf virus

Cas9:

CRISPR-associated endonuclease 9

CDA:

Cytidine deaminase

CRISPR:

Clustered regularly interspaced short palindromic repeats

DSB:

Double strand break

GABA:

γ-Aminobutyric acid

Indel:

Insertion and/or deletion

Rep:

Replication

LIR:

Long intergenic region

sgRNA:

Single guide RNA

SlHWS:

Solanum lycopersicum HAWAIIAN SKIRT

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Acknowledgements

We thank Ms. Kazuko Ito and Ms. Yuri Nemoto at Tsukuba-Plant Innovation Research Center (T-PIRC), University of Tsukuba for technical support. Tomato seeds (cv. Micro-Tom, TOMJPF00001) were provided from T-PIRC, University of Tsukuba through the National Bio-Resource Project (NBRP) of the Japan Agency for Research and Development (AMED), Japan. This work was supported by the Cross-ministerial Strategic Innovation Promotion Program (SIP), Japan, Program on Open Innovation Platform with Enterprises, Research Institute and Academia, Japan Science and Technology Agency (JST, OPERA, JPMJOP1851), and a Cooperative Research Grant from the Plant Transgenic Design Initiative, Gene Research Center, T-PIRC, University of Tsukuba.

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TA, HE, and KM conceived and designed the experiments; KN and AK provided materials and contributed to interpretation of data. SY, IMYA, and SK conducted the experiments and collected data. SY, IMYA, TA, and KM wrote the initial draft of the manuscript. All authors critically reviewed the manuscript and approved the final version of the manuscript.

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Correspondence to Kenji Miura.

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Communicated by Hiroyasu Ebinuma.

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Yuan, S., Kawasaki, S., Abdellatif, I.M.Y. et al. Efficient base editing in tomato using a highly expressed transient system. Plant Cell Rep (2021). https://doi.org/10.1007/s00299-021-02662-z

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Keywords

  • Transient expression
  • Tsukuba system
  • Target-AID
  • Tomato
  • Solanum lycopersicum