Next-generation metabolic engineering offers exciting possibilities for utilizing plants as biofactories to mass-produce valuable metabolites. The CRISPR/Cas9 system has emerged as the most widely adopted genome editing tool for metabolic engineering and crop improvement. However, not all single-guide RNAs (sgRNAs) are equally efficient, and it is crucial to minimize the risk of generating ineffective sgRNAs that could lead to undesired mutants. In this study, we designed and validated the efficiency of two sgRNAs targeting the terminal step of phytate biosynthesis, namely GmIPK1-exon 6 (KS1) and exon 1 (KS2), using various in silico tools.
To assess the functional efficiency of the sgRNAs, we employed AGRODATE (Agrobacterium-mediated disc assay for transient expression) prior to developing stable mutants. Statistical analysis of mutation rates in transient mutants revealed that KS1_sgRNA1 induced deletions (76.4%) ranging from 1 to 7 nucleotides, while insertions accounted for 23.4%. Similarly, KS2_sgRNA2 resulted in 85.2% deletions ranging from 1 to 6 nucleotides, with insertions accounting for 13.2%. Sequence analysis of amplified products confirmed the presence of mutations in 12 out of 16 positive transgenic lines (75%).
Phytate analysis of sgRNA1 knock-out mutants exhibited a remarkable 6.6-fold reduction, while sgRNA2 knock-out mutants showed a substantial 7.05-fold reduction in T0 stable soybean mutants. This research represents the first highly effective application of the CRISPR/Cas9 modification system using chimeric gRNA in the DS9712 soybean cultivar. The findings highlight the significance of developing low phytate soybean variants with immense potential for the food and feed industry.