Articles
A CRISPR/Cas9 approach to improve the quality of tomato by reducing the content of allergens
Article number
1454_5
Pages
35 – 40
Language
English
Abstract
The tomato (Solanum lycopersicum L.) is one of the most important crops for human nutrition and is rich of bioactive compounds that are beneficial for human health, such as antioxidants, iron (Fe), calcium (Ca), vitamins, polyphenols and carotenoids.
However, it can also cause allergies in some consumers, with an estimated incidence ranging from 1.7 to 9.3% in different European populations.
The aim of the research was to reduce the amount of some allergenic proteins in tomato by applying the genome editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9). The targeted genes were Solyc08 g080640, which encode the NP-24 protein, belonging to the thaumatin-like protein (TLP) family, and Solyc10 g080210, which encode the enzyme polygalacturonase 2a (PG2a). Gene-specific sgRNAs with low off-target scores were designed and constructed.
The DNA constructs were assembled using the Goldenbraid system, and the components of the CRISPR-Cas9 system were delivered into plant cells via Agrobacterium-mediated transformation.
Transformation was carried out on Moneymaker plants previously modified for two of the major tomato allergens, the GAME4 gene involved in the biosynthetic pathway of glycoalkaloids and the Sola I 4 gene.
Plants with the desired modifications were identified by genotyping using polymerase chain reaction (PCR) genotyping and confirmed by sequencing the genomic regions surrounding the target sites.
The experiments resulted in a total of eight plants modified for the TLP and PG2a genes with the CRISPR/Cas9 system.
These transformed plants were multiplied in vitro and transferred to the greenhouse to obtain seeds (T0 generation). Lines derived from these modified initial plants, representing the T1 generation, were obtained and characterized by sequencing around the modified sites.
However, it can also cause allergies in some consumers, with an estimated incidence ranging from 1.7 to 9.3% in different European populations.
The aim of the research was to reduce the amount of some allergenic proteins in tomato by applying the genome editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9). The targeted genes were Solyc08 g080640, which encode the NP-24 protein, belonging to the thaumatin-like protein (TLP) family, and Solyc10 g080210, which encode the enzyme polygalacturonase 2a (PG2a). Gene-specific sgRNAs with low off-target scores were designed and constructed.
The DNA constructs were assembled using the Goldenbraid system, and the components of the CRISPR-Cas9 system were delivered into plant cells via Agrobacterium-mediated transformation.
Transformation was carried out on Moneymaker plants previously modified for two of the major tomato allergens, the GAME4 gene involved in the biosynthetic pathway of glycoalkaloids and the Sola I 4 gene.
Plants with the desired modifications were identified by genotyping using polymerase chain reaction (PCR) genotyping and confirmed by sequencing the genomic regions surrounding the target sites.
The experiments resulted in a total of eight plants modified for the TLP and PG2a genes with the CRISPR/Cas9 system.
These transformed plants were multiplied in vitro and transferred to the greenhouse to obtain seeds (T0 generation). Lines derived from these modified initial plants, representing the T1 generation, were obtained and characterized by sequencing around the modified sites.
Authors
C. Montemurro, M. Dellino, M.M. Miazzi, N. Mondelli, S. Presa, D. Orzaez, C. De Giovanni, V. Fanelli, A. Fiore, F. Sevi, G. Diretto, A. Granell
Keywords
tomato allergens, CRISPR/Cas9, thaumatin-like proteins (TLPs), polygalacturonase 2a (PG2a), Goldenbraid
Groups involved
- Division Plant Genetic Resources, Breeding and Biotechnology
- Working Group Genetic Transformation and Gene Editing
- Working Group Horticultural Biotechnology and Breeding
- Division Ornamental Plants
- Division Vegetables, Roots and Tubers
- Division Temperate Tree Fruits
- Division Vine and Berry Fruits
- Division Horticulture for Development
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