Mutation of Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds.

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. encodes the inositol 1,3,4,5,6-pentaphosphate 2-kinase enzyme, which converts -inopsitol-1,3,4,5,6-pentaphosphate (IP) to -inositol-1,2,3,4,5,6-hexakisphosphate (IP) in soybean ( L.). In this study, for developing soybean with low PA levels, we attempted to edit the using the CRISPR/ system to mutations into the gene with guide RNAs in soybean (cv. Kwangankong). The gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean gene-edited lines were obtained in the T generation at editing frequencies of 0.1-84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1-9 nucleotides and insertions of 1 nucleotide in several soybean lines (T). Finally, we confirmed two sgRNA-4 gene-edited homozygote soybean T plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean gene-edited lines indicated a reduction in PA content in soybean T seeds but did not show any defects in plant growth and seed development.