CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds

Abstract

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

Identification and Expression Analysis of the Genes Involved in the Raffinose Family Oligosaccharides Pathway of Phaseolus vulgaris and Glycine max

“…Indeed, duplication and speciation events can lead to the formation of genes with novel functionalities or different expression patterns. However, the high homology between the newly identified genes indicates a high probability that these genes still have the same function which is supported by the research done in G. max [14,45,57]. When the expression patterns are compared in G. max cv.…”

“…At this stage, both genes are expressed more in soybean in comparison with the other galactinol- and RFO synthase genes indicating that they have a possible significant role during seed development. Indeed, their involvement in the RFO biosynthesis pathway during seed development in soybean is confirmed by Le et al (2020) [ 45 ]. Knocking out GmGolS1_A in G. max resulted in a significant reduction of the total RFO content in the seed, whereas the double knockout line of both GmGolS1_A and GmGolS1_B further reduced the total RFO content but to a lesser extent.…”

“…Our study confirms the results of the earlier identified genes involved in the RFO pathway. Le et al (2020) identified the same six GolS genes and showed that GmGolS1_A and GmGolS2_B both contribute to the production of RFOs through two CRISPR/Cas9-mediated knockout lines [ 45 ]. Dierking and Bilyeu (2008) identified the same three RS genes and Valentine et al (2017) proved that GmRS2 contributes to the production of raffinose with the use of a soybean line in which GmRS2 was silenced [ 7 , 14 ].…”

“…Targeting this gene could potentially lead to unwanted phenotypic side effects. However, this does not necessarily have to be the case as was observed in a soybean double mutant where both the GolS1 genes were knocked out, with no adverse effects on the plants’ growth [ 45 ]. Besides the formation of stachyose, SS also facilitates the formation of galactinol cyclitols which additionally contribute to the digestive problems associated with RFOs [ 37 , 42 ].…”

“…Valentine et al (2017) reduced the raffinose and stachyose content in seeds from respectively, 0.63% and 3.79% in the wild type to 0.11% and 1.21% in a transgenic line using a silencing construct targeting a RS isoform [ 14 ]. In a recent study by Le et al (2020) two GolS genes were knocked out, reducing the total RFO content by 35.2% [ 45 ]. However, more progress can be made to lower the RFO content in the seeds more efficiently without compromising normal plant development and functioning.…”