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Wheat \u03b1\u2010gliadins are a major contributor to the autoimmune response, as they contain one of the most immunogenic peptides, the 33\u2010mer, along with numerous variants. In this study, we used CRISPR\/Cas technology to mutate genes encoding \u03b1\u2010gliadins. This approach employed paired sgRNAs to precisely excise immunogenic regions while preserving non\u2010immunogenic sequences within the \u03b1\u2010gliadins. Furthermore, we replaced the excised region with an \u03b1\u2010gliadin\u2010based double\u2010stranded oligodeoxynucleotide (dsODN) designed with nucleotide changes to reduce immunoreactivity and increase peptidase cleavage sites. Two different CRISPR systems, Cas9 and Cas12a, were applied to generate wheat protoplasts and plants with non\u2010immunogenic regions. Cas9 demonstrated superior performance in terms of editing frequency, excision and replacement of immunogenic fragments. However, the Cas12a nuclease (Cpf1) showed promising editing efficiency, offering the potential for future wheat editing applications. Using the Cas9 system, we achieved a 74.2% excision rate of the 33\u2010mer in wheat plants. Subsequent analyses showed a significant reduction in the reactivity to the G12 monoclonal antibody, capable of identifying the 33\u2010mer peptide and a decrease in the prolamin levels compared to the wild\u2010type. Additionally, we developed a high\u2010throughput sequencing\u2010based software specifically designed to identify mutations in multi\u2010copy gene families. This innovative tool enabled fast, parallel screening of the samples in this study and facilitated the identification of the specific editing patterns produced by the designed constructs.<\/jats:p>","DOI":"10.1111\/pbi.70200","type":"journal-article","created":{"date-parts":[[2025,6,15]],"date-time":"2025-06-15T13:10:02Z","timestamp":1749993002000},"page":"3798-3813","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Cas9\u2010 and Cas12a\u2010mediated excision and replacement of the celiac disease\u2010related \u03b1\u2010gliadin immunogenic complex in hexaploid wheat"],"prefix":"10.1111","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0040-1053","authenticated-orcid":false,"given":"Miriam","family":"Mar\u00edn\u2010Sanz","sequence":"first","affiliation":[{"name":"Department of Plant Breeding Institute for Sustainable Agriculture (IAS\u2010CSIC)  C\u00f3rdoba Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susana","family":"S\u00e1nchez\u2010Le\u00f3n","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding Institute for Sustainable Agriculture (IAS\u2010CSIC)  C\u00f3rdoba Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda H.","family":"Guzm\u00e1n\u2010L\u00f3pez","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding Institute for Sustainable Agriculture (IAS\u2010CSIC)  C\u00f3rdoba Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Colby G.","family":"Starker","sequence":"additional","affiliation":[{"name":"Department of Genetics, Cell Biology and Development, Center for Precision Plant Genomics University of Minnesota  St. Paul MN USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4944-1224","authenticated-orcid":false,"given":"Daniel F.","family":"Voytas","sequence":"additional","affiliation":[{"name":"Department of Genetics, Cell Biology and Development, Center for Precision Plant Genomics University of Minnesota  St. Paul MN USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7652-229X","authenticated-orcid":false,"given":"Francisco","family":"Barro","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding Institute for Sustainable Agriculture (IAS\u2010CSIC)  C\u00f3rdoba Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,6,15]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1136\/gut.2005.064550"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2015.06.427"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-021-01133-w"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aar7191"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1053\/gast.2002.35381"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.3389\/fpls.2020.584151"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tig.2016.06.007"},{"key":"e_1_2_10_9_1","volume-title":"BBMap: a Fast, Accurate, Splice\u2010Aware Aligner","author":"Bushnell B.","year":"2014"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12916-019-1380-z"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1094\/CCHEM.1999.76.6.907"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1105\/tpc.16.00922"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-arplant-050718-100049"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-019-0032-3"},{"key":"e_1_2_10_15_1","first-page":"1","article-title":"An optimised CRISPR\/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat","volume":"15","author":"Cui X.","year":"2019","journal-title":"Plant Methods"},{"key":"e_1_2_10_16_1","volume-title":"Usearch","author":"Edgar R.","year":"2010"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/ni.1780"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1007773107"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcs.2012.02.009"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0090898"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0091931"},{"key":"e_1_2_10_22_1","first-page":"411","article-title":"Dietary Protein Processing: Intraluminal and Enterocyte Surface Events. 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