Fig 1: Evidence for knockout of the Kunitz trypsin inhibitor protein. (A–B) Confirmation of Kunitz trypsin inhibitor protein reduction by Western blot analysis using a Kunitz trypsin inhibitor (KTI) antibody in conventionally bred triple‐mutant lines (A) and clustered regularly interspaced short palindromic repeats (CRISPR)‐derived triple‐mutant lines (B), respectively. MN0811CN‐BC4TN is derived from MN0811CN; M07‐292111‐BC4TN2 and M07‐292111‐BC4TN1 are derived from M07‐292111. WPT673‐7‐12‐5 and WPT673‐7‐8‐8 (where WPT is whole plant transformation) are edited lines developed in the Bert background. (C–D) Trypsin inhibition activity in conventionally bred triple‐mutant lines (C) and CRISPR‐derived triple‐mutant lines (D), respectively. Black asterisks in (C) correspond to recurrent parent MN0811CN, while blue asterisks correspond to recurrent parent M07‐292111. Shown is the average of three technical replicates, and error bars indicate SD. **p < 0.001, ***p < 0.001, ****p < 0.0001, adjusted p‐value for one‐way analysis of variance (ANOVA) (C–D). (E–F) Confirmation of KTi3 protein reduction in total seed proteome by dia‐parallel accumulation–serial fragmentation (PASEF)‐based proteomics analysis. (E) Each genotype value represents the average of two technical replicates of ∼100 bulk seeds from 20–25 field‐harvested plants per genotype. (F) Each genotype value represents the average of three technical replicates of ∼100 bulk seeds from 20–25 field‐harvested plants per genotype. Error bars indicate SD. **p < 0.001, ***p < 0.001, ****p < 0.0001, adjusted p‐value Tukey t‐test.