{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T02:21:00Z","timestamp":1789698060058,"version":"4.0.1"},"reference-count":55,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T00:00:00Z","timestamp":1773014400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Plant Sci."],"abstract":"<jats:p>\n                    Gly m 4 is a major allergen in soy milk and cross-reacts with Bet v 1, the primary birch pollen allergen. To reduce Gly m 4 allergenicity, we performed site-directed mutagenesis of the\n                    <jats:italic>Gly m 4<\/jats:italic>\n                    gene in soybean through a DNA-free\n                    <jats:italic>in planta<\/jats:italic>\n                    bombardment method using ribonucleoproteins (iPB-RNP). Although a mutant line (\n                    <jats:italic>\n                      Gly m 4-2\n                      <jats:sup>del<\/jats:sup>\n                    <\/jats:italic>\n                    ) was generated by targeting the\n                    <jats:italic>Gly m 4-2<\/jats:italic>\n                    gene, immunoblot analysis revealed that translation products from other homologs still accumulated in the seed tissues. Subsequent gene expression analysis identified\n                    <jats:italic>Gly m 4-L1<\/jats:italic>\n                    , one of eight\n                    <jats:italic>Gly m 4<\/jats:italic>\n                    homologs in the soybean genome, as the primary target for further mutagenesis. Ribonucleoprotein complexes loaded with either a single guide RNA (gRNA) or two distinct gRNAs targeting\n                    <jats:italic>Gly m 4-L1<\/jats:italic>\n                    were introduced into the shoot apical meristems. Sequencing analysis identified three mutant lines: an 8-bp deletion (\n                    <jats:italic>\n                      Gly m 4-L1\n                      <jats:sup>8-del<\/jats:sup>\n                    <\/jats:italic>\n                    ), a 128-bp insertion (\n                    <jats:italic>\n                      Gly m 4-L1\n                      <jats:sup>128-ins<\/jats:sup>\n                    <\/jats:italic>\n                    ), and a complete gene deletion (\n                    <jats:italic>\n                      Gly m 4-L1\n                      <jats:sup>null<\/jats:sup>\n                    <\/jats:italic>\n                    ). Immunoblot analysis confirmed the absence of Gly m 4 protein accumulation in the seeds of these mutants. To evaluate immunoglobulin E (IgE) reactivity, protein extracts from\n                    <jats:italic>\n                      Gly m 4-2\n                      <jats:sup>del<\/jats:sup>\n                    <\/jats:italic>\n                    ,\n                    <jats:italic>\n                      Gly m 4-L1\n                      <jats:sup>null<\/jats:sup>\n                    <\/jats:italic>\n                    , and wild-type plants were incubated with sera from patients positive for Gly m 4\u2013specific IgE. Protein extracts from the\n                    <jats:italic>\n                      Gly m 4-L1\n                      <jats:sup>null<\/jats:sup>\n                    <\/jats:italic>\n                    line showed markedly reduced IgE binding compared with\n                    <jats:italic>\n                      Gly m 4-2\n                      <jats:sup>del<\/jats:sup>\n                    <\/jats:italic>\n                    and wild-type samples. These findings demonstrate that\n                    <jats:italic>Gly m 4-L1<\/jats:italic>\n                    is the key gene responsible for Gly m 4 allergen production in soybean seeds.\n                  <\/jats:p>","DOI":"10.3389\/fpls.2026.1739979","type":"journal-article","created":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T06:46:09Z","timestamp":1773038769000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["CRISPR\/Cas9 ribonucleoprotein-mediated knockout of Gly m 4-L1 eliminates allergen accumulation in soybean"],"prefix":"10.3389","volume":"17","author":[{"given":"Shinya","family":"Chatani","sequence":"first","affiliation":[{"name":"Graduate School of Agriculture, Hokkaido University","place":["Hokkaido, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chikako","family":"Kuwabara","sequence":"additional","affiliation":[{"name":"Graduate School of Agriculture, Hokkaido University","place":["Hokkaido, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miki","family":"Hibara","sequence":"additional","affiliation":[{"name":"Graduate School of Agriculture, Hokkaido University","place":["Hokkaido, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Kazahaya","sequence":"additional","affiliation":[{"name":"Graduate School of Agriculture, Kyoto University","place":["Kyoto, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayaka","family":"Tange","sequence":"additional","affiliation":[{"name":"Graduate School of Agriculture, Kyoto University","place":["Kyoto, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuichiro","family":"Tamamoto","sequence":"additional","affiliation":[{"name":"Graduate School of Agriculture, Hokkaido University","place":["Hokkaido, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshiyuki","family":"Hirata","sequence":"additional","affiliation":[{"name":"Field Science Center Northern Biosphere, Hokkaido University","place":["Hokkaido, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuma","family":"Fukutomi","sequence":"additional","affiliation":[{"name":"NHO Sagamihara National Hospital","place":["Kanagawa, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuyuki","family":"Maruyama","sequence":"additional","affiliation":[{"name":"Graduate School of Agriculture, Kyoto University","place":["Kyoto, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tetsuya","family":"Yamada","sequence":"additional","affiliation":[{"name":"Graduate School of Agriculture, Hokkaido University","place":["Hokkaido, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,3,9]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1007\/s11248-020-00229-4","article-title":"Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation","volume":"30","author":"Adachi","year":"2021","journal-title":"Transgenic Res."},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1016\/j.afres.2022.100154","article-title":"Plant-based meat alternatives: Compositional analysis, current development and challenges","volume":"2","author":"Ahmad","year":"2022","journal-title":"Appl. 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