Bioscience Evidence 2025, Vol.15, No.5, 228-236 http://bioscipublisher.com/index.php/be 230 volume and texture of the bread made are also not good. Therefore, conventional breeding needs to strike a balance between reducing immunogenicity and maintaining the integrity of the gluten network, and also meet the needs of the food industry. 3.3 Use of natural genetic variation within wheat and related species Wheat and its related species have rich natural differences in gluten composition and the number of immunogenic epitopes. Studies have shown that the immunogenicity of α- and γ -olysis proteins in durum wheat, some wild species and hybrids (such as the wheat-rye hybrid tritordeum) is lower than that in common wheat (Ozuna and Barro, 2018; Pilolli et al., 2019; Marin-Sanz et al., 2023). These genotypes can be used as parents for breeding, and common varieties can be introduced through hybridization or backcrossing, thereby gradually reducing the immunogenicity of gluten. In addition, directed deletion of chromosomal fragments or the use of chromosomal translocations (such as 1BL/1RS rye translocations) can also regulate gluten composition, providing more means for breeding (Pilolli et al., 2019; Marin-Sanz et al., 2023). 4 Modern Genetic Approaches 4.1 RNA interference (RNAi) RNAi technology can silence some gluten-related genes, thereby significantly reducing the expression of α-, γ- and ω -alcohol-soluble proteins. Studies have found that after RNAi treatment, the gluten content in wheat grains can decrease to 97%, and the stimulating effect of this flour on T cells in patients with celiac disease is significantly weakened, with little impact on dough quality (Altenbach et al., 2019; Garcia-Molina et al., 2019; Jouanin et al., 2019; Sharma et al., 2020). However, because the builders of RNAi remain in the genome, this type of wheat is often identified as genetically modified crops and is subject to regulatory restrictions (Jouanin et al., 2019; Sharma et al., 2020). 4.2 CRISPR/Cas genome editing CRISPR/Cas9 technology can precisely knock out or modify multiple gluten genes, such as α- and γ -glycolin. Many studies have obtained wheat mutants of low trough protein, whose immune reactivity has decreased by approximately 85%, and some strains have no transgenic residue (Sanchet-leon et al., 2017; Jouanin, 2019; Jouanin et al., 2019; 2020; Liu et al., 2023; Pourmohammadi et al., 2023; Bennur et al., 2024) (Figure 1). CRISPR/Cas9 can also replace some immunogenic epitopes with "safe" epitopes, which can not only improve the quality of bread but also reduce the risk of celiac disease (Jouanin et al., 2020; Liu et al., 2023). However, if gluten is completely knocked out, it may affect the processing performance of the dough. Therefore, a balance needs to be struck between safety and quality (Jouanin et al., 2019; 2020; Liu et al., 2023). 4.3 TILLING (targeting induced local lesions in genomes) TILLING is a non-transgenic mutagenesis method that uses mutagenesis and then screens to identify mutants of the target gene. TILLING obtained some wheat materials with lower gluten content and weakened immunogenicity on genes such as DEMETER and DRE2 that regulate gluten. These materials can also retain high-molecular-weight gluten subunits, so the processing performance of the bread is basically maintained (Moehs et al., 2019; Wen et al., 2022). However, some mutants may present problems such as decreased pollen viability, which still require further improvement (Wen et al., 2022). 4.4 Transgenic approaches Transgenic methods involve adjusting the composition of gluten by introducing or silencing specific genes. For instance, introducing wild-type genes into mutants can restore the original phenotype. Alternatively, silencing certain protein subunits with RNAi can also reduce immunogenicity (Vasil and Anderson, 1997; Altenbach et al., 2019; Moehs et al., 2019). Although this method works well in the laboratory, it is limited by public acceptance and policy regulations in practical promotion (Vasil and Anderson, 1997; Altenbach et al., 2019). 5 Case Study: CRISPR/Cas9 Editing of Gliadin Genes in Bread Wheat 5.1 Research background: α-gliadins as key immunogenic proteins The α-gliadins in wheat gluten contain many immune epitopes related to Celiac disease (CD) and are the main
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