The GE-high GABA tomato line (#87-17) was generated by editing the glutamic acid decarboxylase gene (gad) of Solanum lycopersicum variety Sicilian Rouge CF using the CRISPR/Cas9 technology. Aim of the mutation was to enhance γ-aminobutyric acid (GABA) productivity in tomato fruit. GABA is a non-proteinogenic amino acid expected to provide various benefit to human health, such as lowering blood pressure.
Glutamate decarboxylase (GAD) is a key enzyme in GABA biosynthesis. It has a C-terminal autoinhibitory domain (known as calmodulin (CaM)-binding domain (CaMBD)) that regulates enzymatic function. The loss of this domain increases GAD activity.
The tomato genome contains five gad genes of which Slgad1, Slgad2 and Slgad3 are expressed during fruit development. The developer targeted the Slgad3 gene (Solyc01g005000) that plays a major role in GABA accumulation in fruits.
The line was developed by Agrobacterium tumefaciens mediated transformation with T-DNA containing the coding sequence for Cas9 and the DNA sequences for target-gene-specific small guide RNAs. Null segregant lines were obtained by self-crossing and line #87-17 was selected based on seed number and GABA content.
The commercialized line #87-17 showed a 1 bp insertion at the targeted position which induces a stop codon immediately upstream of the autoinhibitory domain in the C-terminus.
This line showed 4 to 6 times higher GABA accumulation in red-stage tomato fruits. The trait was shown to be stable through at least three generations.
In a former work, the developer studied the impact of editing the Slgad2 and Slgad3 genes in several tomato lines (Nonaka et al. (2017), Sci Rep. 7:7057).
Source:
USDA-APHIS letter of inquiry 20-140-01_air
MAFF, notification of the Director-General of the Consumption and Safety Bureau, Ministry of Agriculture, Forestry and Fisheries (translation of the document using DeepL).