Mutation of <i>GmIPK1</i> Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds

Mutation of <i>GmIPK1</i> 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. <i>GmIPK1</i> encodes the inositol 1,3,4,5,6-penta<i>kis</i>phosphate 2-kinase enzyme, which converts...

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Bibliographic Details
Main Authors: Ji Hyeon Song, Gilok Shin, Hye Jeong Kim, Saet Buyl Lee, Ju Yeon Moon, Jae Cheol Jeong, Hong-Kyu Choi, In Ah Kim, Hyeon Jin Song, Cha Young Kim, Young-Soo Chung
Format: Article
Language:English
Published: MDPI AG 2022-09-01
Series:International Journal of Molecular Sciences
Subjects:
soybean
phytic acid
genome editing
CRISPR/Cas9
sgRNA
<i>GmIPK1</i>
Online Access:https://www.mdpi.com/1422-0067/23/18/10583
Description
Summary: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. <i>GmIPK1</i> encodes the inositol 1,3,4,5,6-penta<i>kis</i>phosphate 2-kinase enzyme, which converts <i>myo</i>-inopsitol-1,3,4,5,6-penta<i>kis</i>phosphate (IP<sub>5</sub>) to <i>myo</i>-inositol-1,2,3,4,5,6-hexakisphosphate (IP<sub>6</sub>) in soybean (<i>Glycine max</i> L.). In this study, for developing soybean with low PA levels, we attempted to edit the <i>GmIPK1 <i>gene</i></i> using the CRISPR/<i>Cas9</i> system to <i>introduce</i> mutations into the <i>GmIPK1</i> gene with guide RNAs in soybean (cv. Kwangankong). The <i>GmIPK1</i> gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean <i>Gmipk1</i> gene-edited lines were obtained in the T<sub>0</sub> 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<sub>0</sub>). Finally, we confirmed two sgRNA-4 <i>Gmipk1</i> gene-edited homozygote soybean T<sub>1</sub> plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean <i>Gmipk1</i> gene-edited lines indicated a reduction in PA content in soybean T<sub>2</sub> seeds but did not show any defects in plant growth and seed development.
ISSN:1661-6596
1422-0067

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