Targeted editing of tomato carotenoid isomerase reveals the role of 5′ UTR region in gene expression regulation

Abstract

Key Message

A deletion created by CRISPR/Cas9 system in the 5’ UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene.

Abstract

CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5′ UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5′ UTR region lowers the expression of the gene leading to changes in plant phenotype.

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Data availability

The RNA-Seq data have been submitted to NCBI and is available under the bioproject PRJNA639003.

Abbreviations

CRISPR/Cas:

Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR associated protein

TILLING:

Targeting Induced Local Lesions in Genomes

UTR:

Untranslated region

CRTISO:

Carotenoid isomerase

LCYB:

Lycopene-β-Cyclase

qPCR:

Quantitative PCR

WT:

Wild type

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Acknowledgments

The authors acknowledge the financial and research material support from SciGenom Research Foundation. They also thank Dr. N. S. Kalesh and team, Interfield Laboratories, Kochi, India for their help and technical advice in the HPLC analysis of the fruit pigments, OmicsGen Life Sciences, Kochi for support in carrying out quantitative RT-PCR and the technical advice on tomato transformation from Prof. M. V. Rajam, Department of Genetics, University of Delhi, South Campus, New Delhi, India. The authors acknowledge the advice from the Institutional Biosafety Committee of AgriGenome Labs Private Limited, Kochi, India. We also thank Mr. Suhail. A, AgriGenome Labs Pvt. Ltd., for the help in the preparation of the graphics and Dr. Sameer Phalke, SciGenom Labs for reading the manuscript and providing valuable suggestions.

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NT, SSE and BK conceptualized the study; BK, RA and SN designed the experiments; AA and SA made the vector constructs; NT and UMM carried out the plant transformations; LJK, MJ, SSU carried out the molecular and biochemical analysis; LJK, VBR, NC and BK carried out the qPCR and RNAseq data analysis; BK and AA wrote the manuscript; BK, GT, SSE and SN supervised the studies; SSE and GT provided resources and funding.

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Correspondence to Boney Kuriakose.

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Communicated by Yiping Qi.

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Lakshmi Jayaraj, K., Thulasidharan, N., Antony, A. et al. Targeted editing of tomato carotenoid isomerase reveals the role of 5′ UTR region in gene expression regulation. Plant Cell Rep (2021). https://doi.org/10.1007/s00299-020-02659-0

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Keywords

  • CRISPR
  • Cas9
  • Tomato
  • Genome editing
  • CRTISO
  • Carotenoid
  • Lycopene
  • Tangerine