crispr/cas9-targeted Mutagenesis of KTI1 and KTI3 to Reduce Trypsin Inhibitors in Soybean Seeds

Trypsin inhibitor (TI) in soybean seeds, restrains the function of trypsin, causing low protein digestibility when raw soybeans are fed to animals. Heat treatment has been widely used to deactivate TI, but it is energy-intensive and costly, and degrades protein quality. Despite a few soybean accessions harboring natural low TI content have been identified, multiple TI genes and lacking of gene-based markers still hinder the breeding success of low TI soybean cultivars. The objectives of this study were to concisely edit the major genes contributing to the TI content and activity specifically in the soybean seeds using CRISPR/Cas9 system, and develop allele-specific molecular markers based on the generated mutant alleles. With the aid of TI gene expression data and real-time PCR results, KTI1 (Glyma01g095000) and KTI3 (Glyma08g341500), were selected as the target genes. Then, we developed a productive CRISPR/Cas9 construct for the transformation on Glycine max cv. Williams 82. (WM82). The results showed that in the seeds at T0 generation, the gene editing has been all complete for KTI1 while it has been partly finished for KTI3. Consistent with genotyping results, the TI content and activity in gene edited seeds declined 70% and 10% with knock-out of KTI1 alone and 90% and 30% with knock-out of both KTI1 and KTI3, which were also both lower than that in the seeds of PI 547656, the natural low TI soybean accession. Furthermore, in T1 seeds, we collected one transgene free line #5-26 with double homozygous mutations. Based on the mutant alleles in #5-26, we developed molecular markers to effectively screen the mutant alleles of KTI1 and KTI3 for the perspective breeding of low TI soybean varieties. The soybean line and selection markers acquired from this study will assist in accelerating the introduction of low TI trait to elite soybean cultivars with added-value.