abstract
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Bread wheat (Triticum aestivum L.) is consumed worldwide, and 20% of our calorie intake is from bread wheat. In addition, wheat with highly functional ingredients is directly beneficial for human health. The γ-aminobutyric acid (GABA) is one of the highly functional ingredients and have effects such as lowering of blood pressure and mental stability among patients with lifestyle related diseases, namely, high blood pressure. Recently, tomatoes with high GABA content have been produced by CRISPR/Cas9 genome editing and are already in the market. In plants, the biosynthesis of GABA is catalyzed by glutamate decarboxylase (GAD), which has a C-terminal autoinhibitory domain. Moreover, deletion of this domain leads to an increased GAD activity and therefore increased GABA content in tomato and rice. In this study, a genome editing approach was applied to the GAD genes in bread wheat cultivar Fielder to increase the GABA content in wheat seeds. We found 19 GAD genes in Fielder genome. Among them, TaGAD1_B and TaGAD1_D were selected as the target for genome editing using CRISPR/Cas9 because upregulation of these genes in the seed leads to an increase in the GABA content, as revealed in our previous study. Deduced amino acids sequence confirms that TaGAD1 has a C-terminal autoinhibitory domain. According to the calmodulin binding assay, the domain of TaGAD1 showed calmodulin binding activity; therefore, an increased GAD activity is expected upon deletion of this domain. Common targets of gRNA were designed for TaGAD1_B and TaGAD1_D. The gRNA and Cas9 expression vectors were constructed separately: pTaU6gRNA and pE (R4-R3) ZmUbi_OsCas9_ver.3, respectively. Afterward, each plasmid DNA was delivered into the immature embryos by particle bombardment. To evaluate the mutation, genomic DNA was extracted from the embryos 1 week after the bombardment, followed by PCR and digestion with the restriction enzyme. The mutation efficiency was found to be 3.6%. However, confirming the sequences, all mutants detected on the target site were only 1-bp substitution, which does not disrupt the activity of the autoinhibition domain. In future, to increase TaGAD1 activity in wheat, redesigning of the gRNA target is needed.