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Background  Gamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things. GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). In plants the enzymatic activity of GAD is activated by Ca 2+ /calmodulin binding (CaMBD) at the C-terminus in response to various stresses, allowing rapid GABA accumulation in cells. GABA plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. Furthermore, it is known to be a health-promoting functional substance that exerts improvements in life-style related diseases such as hypertension, diabetes, hyperlipidemia, and so on. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Therefore, we attempted a genetic modification of rice  GAD  via genome editing technology to increase GABA levels in the edible part of rice.    Results  In this study, we focused on  GAD3 , one of five  GAD  genes present in the rice genome, because  GAD3  is the predominantly expressed in seeds, as reported previously. We confirmed that GAD3 has an authentic Ca 2+ /CaMBD that functions as an autoinhibitory domain. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD off from the  OsGAD3  gene, then introducing this transgene into rice scutellum-derived calli using an all-in-one vector harboring guide RNAs and CRISPR/Cas9 via  Agrobacterium  to regenerate rice plants. Out of 24 transformed rice (T 1 ), a genome-edited rice line (#8_8) derived from two independent cleavages and ligations in the N-terminal position encoding  Os GAD3-CaMBD and 40 bp downstream of the termination codon, respectively, displayed a  AK NQDAAD peptide in the C-terminal region of the putative  Os GAD3 in place of its intact CaMBD (bold indicates the trace of the N-terminal dipeptides of the authentic CaMBD). A very similar rice line (#8_1) carrying  AK NRSSRRSGR in  Os GAD3 was obtained from one base pair deletion in the N-terminal coding region of the CaMBD. Free amino acid analysis of the seeds (T 2 ) indicated that the former line contained seven-fold higher levels of GABA than wild-type, whereas the latter line had similar levels to the wild-type, although in vitro enzyme activities of recombinant GAD proteins based on the GAD3 amino acid sequence elucidated from these two lines in the absence of Ca 2+ /bovine CaM were both higher than wild-type counterpart. In addition to high level of GABA in #8_8, the average seed weight per grain and protein content were superior to wild-type and #8_1.    Conclusions  We have successfully established GABA-fortified rice by using CRISPR/Cas9 genome editing technology. Modified rice contained seven-fold higher GABA content and furthermore displayed significantly higher grain weight and protein content than wild-type brown rice. This is the first report of the production of GABA-enriched rice via a genome editing.

rice

An In Vivo Targeted Deletion of the Calmodulin-Binding Domain from Rice Glutamate Decarboxylase 3 (OsGAD3) Increases γ-Aminobutyric Acid Content in Grains