Site-directed mutagenesis via gene targeting (GT) based on homologous recombination is the ultimate mutation breeding technology because it enables useful information acquired from structural-and computational-based protein engineering to be applied directly to molecular breeding, including metabolic engineering, of crops. Here, we employed this rationale to introduce precise mutations in OASA2-an a-subunit of anthranilate synthase that is a key enzyme of tryptophan (Trp) biosynthesis in rice (Oryza sativa)-via GT, with subsequent selection of GT cells using a Trp analog. The expression level of OASA2 in plants homozygous and heterozygous for modified OASA2 was similar to that of nontransformants, suggesting that OASA2 transcription in GT plants was controlled in the same manner as endogenous OASA2, and that GT could lead to a lower risk of gene silencing than in conventional overexpression approaches. Moreover, we showed that enzymatic properties deduced from protein engineering or in vitro analysis could be reproduced in GT plants as evidenced by Trp accumulation levels. Interestingly, mature seeds of homozygous GT plants accumulated Trp levels 230-fold higher than in nontransformants without any apparent morphological or developmental changes. Thus, we have succeeded in producing a novel rice plant of great potential nutritional benefit for both man and livestock that could not have been selected using conventional mutagenesis approaches. Our results demonstrate the effectiveness of directed crop improvement by combining precision mutagenesis via GT with a knowledge of protein engineering.Comparative genomics and structural-and computational-based protein engineering provide copious amounts of useful information that can be applied to crop improvement. In conventional mutation breeding technology using chemical mutagens and ionizing radiation, it remains quite difficult to select mutants harboring target genes modified exactly as required, because mutations occur randomly. Meanwhile, conventional transformation technology based on information from comparative genomics and protein engineering in crops remains a powerful tool in the field of molecular breeding. Agronomical traits such as herbicide tolerance, stress tolerance, and photosynthetic activity have been modified successfully in several crops using conventional transformation technologies that depend on overexpression of modified, highly functional enzymes (Rao, 2008). Moreover, conventional transformation technology is also essential for introducing exogenous genes that do not exist naturally in target plants, e.g. for the production of recombinant proteins. However, mutational approaches are considered better than conventional transformation approaches when introducing directed mutations into endogenous genes to confer novel traits to crops, because conventional transformation technologies cannot eliminate the endogenous targeted gene itself. Thus, a clear goal of current mutation breeding is to modify targeted endogenous genes directly to yield the ...