ORIGINAL RESEARCH article
Front. Plant Sci.
Sec. Plant Biotechnology
Volume 16 - 2025 | doi: 10.3389/fpls.2025.1612747
This article is part of the Research TopicEngineering Future Crops Through Genome EditingView all 15 articles
CRISPR/Cas9-Mediated Simultaneous Targeting of GmP34 and Its Homologs Produces T-DNA-Free Soybean Mutants with Reduced Allergenic Potential
Provisionally accepted
Dongwon Baek1
Byung Jun Jin2Mi Suk Park1Ye Jin Cha2Tae Hee Han2Ye Na Jang2Su Bin Kim2
Sangin Shim3
Jong Il Chung3Hyun Jin Chun3*
Min Chul Kim2*- 1Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Gyeongsang, Republic of Korea
- 2Division of Applied Life Science, Gyeongsang National University, Jinju, Republic of Korea
- 3Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, South Gyeongsang, Republic of Korea
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Soybean (Glycine max L.) P34 (GmP34) is a prominent allergenic seed protein belonging to the papainlike cysteine protease family. To mitigate its allergenic potential, we implemented a CRISPR/Cas9based genome editing strategy targeting GmP34 along with its two highly similar homologs, GmP34h1 and GmP34h2, in the soybean cultivar Williams 82. Phylogenetic analysis and domain characterization identified GmP34h1 and GmP34h2 as the closest homologs to GmP34, with conserved allergenic peptide motifs. Gene expression profiling revealed similar expression patterns of all three genes during seed maturation, indicating potential functional redundancy. Two multiplex CRISPR/Cas9 constructs were designed to simultaneously target GmP34/GmP34h1 and GmP34/GmP34h1/GmP34h2 genes, respectively. Transgenic genome editing plants were generated via Agrobacterium-mediated transformation, and targeted mutagenesis was confirmed by genomic PCR and deep sequencing. Loss of GmP34 protein in edited lines was further validated through western blot analysis. Using this strategy, we successfully generated GmP34 single, GmP34/GmP34h1 double, and GmP34/GmP34h1/GmP34h2 triple mutants. This study highlights the utility of multiplex genome editing in silencing soybean allergenic gene and its homologs. Ongoing analyses of allergenicity in these edited lines aim to provide a genetic foundation for the development of hypoallergenic soybean cultivars through precise genome engineering.
Keywords: Soybean, allergen, CRISPR/Cas9, Genome editing, GmP34 homologs
Received: 16 Apr 2025; Accepted: 18 Jul 2025.
Copyright: © 2025 Baek, Jin, Park, Cha, Han, Jang, Kim, Shim, Chung, Chun and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Hyun Jin Chun, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 660–701, South Gyeongsang, Republic of Korea
Min Chul Kim, Division of Applied Life Science, Gyeongsang National University, Jinju, Republic of Korea
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