Bioscience Evidence 2025, Vol.15, No.5, 228-236 http://bioscipublisher.com/index.php/be 231 proteins that cause immune responses in patients. Because of the large number and complex distribution of α-, γ- and ω -olysis protein genes, it is difficult for traditional breeding to remove all immune epitopes (Jouanin et al., 2019; Jouanin et al., 2020; Guzmán-López et al., 2021) (Figure 2). Figure 1 Breeding of a green super wheat variety through CRISPR/Cas9 (Adopted from Bennur et al., 2024) Figure 2 MUSCLE alignment of Amps clusters from the neighbor-joining dendrogram. (A) Cluster 1 of Amps present in lines with DP genotype. (B) Cluster 1 of Amps present in lines with BW208 genotype. (C) Cluster 22 of Amps present in lines with BW208 genotype. DP: Don Pedro. Amps: unique denoised amplicons (Adopted from Guzmán-López et al., 2021) 5.2 Experimental strategy: multiplex CRISPR editing of gliadin gene families Researchers employed multiple Sgrnas to target the conserved regions of the α-, γ-, and ω -olysin gene families. Through multiple CRISPR/Cas9 editing, they can simultaneously knock out or modify multiple gene copies, thereby significantly reducing the expression of immunogenic epitopes (Jouanin et al., 2019; Guzman Lopez et al., 2021; Yu et al., 2023; Sanchez-Leon et al., 2024).
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