Dataset Information

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.

SUBMITTER: Le H

PROVIDER: S-EPMC7773711 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

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

Le Huy H Nguyen Nhung Hong NH Ta Dong Thị DT Le Thao Nhu Thi TNT Bui Thao Phuong TP Le Ngoc Thu NT Nguyen Cuong Xuan CX Rolletschek Hardy H Stacey Gary G Stacey Minviluz G MG Pham Ngoc Bich NB Do Phat Tien PT Chu Ha Hoang HH

Frontiers in plant science 20201217

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, <i>GmGOLS1A</i> and its homeolog <i>GmGOLS1B</i>. Genotyping of T0 plants showed that the construct design was efficient in ind ...[more]

PMID: 33391326

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Project description:Soybean [Glycine max (L.) Merr.] is an important world economic crop. It is rich in oil, protein, and starch, and soluble carbohydrates in soybean seeds are also important for human and livestock consumption. The predominant soluble carbohydrate in soybean seed is composed of sucrose and raffinose family oligosaccharides (RFOs). Among these carbohydrates, only sucrose can be digested by humans and monogastric animals and is beneficial for metabolizable energy, while RFOs are anti-nutritional factors in diets, usually leading to flatulence and indigestion, ultimately reducing energy efficiency. Hence, breeding efforts to remove RFOs from soybean seeds can increase metabolizable energy and improve nutritional quality. The objective of this research is to use the multiplex Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing system to induce the knockout of soybean raffinose synthase (RS) genes RS2 and RS3 simultaneously to reduce RFOs in mature seeds. First, we constructed five types of multiplex gene editing systems and compared their editing efficiency in soybean hairy roots. We confirmed that the two-component transcriptional unit (TCTU) and single transcriptional unit (STU) systems with transfer RNA (tRNA) as the cleavage site performed better than other systems. The average editing efficiency at the four targets with TCTU-tRNA and STU-tRNA was 50.5% and 46.7%, respectively. Then, we designed four single-guide RNA (sgRNA) targets to induce mutations at RS2 and RS3 by using the TCTU-tRNA system. After the soybean transformation, we obtained several RS2 and RS3 mutation plants, and a subset of alleles was successfully transferred to the progeny. We identified null single and double mutants at the T2 generation and analyzed the seed carbohydrate content of their progeny. The RS2 and RS3 double mutants and the RS2 single mutant exhibited dramatically reduced levels of raffinose and stachyose in mature seeds. Further analysis of the growth and development of these mutants showed that there were no penalties on these phenotypes. Our results indicate that knocking out RS genes by multiplex CRISPR/Cas9-mediated gene editing is an efficient way to reduce RFOs in soybean. This research demonstrates the potential of using elite soybean cultivars to improve the soybean meal trait by multiplex CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-mediated gene editing.

Project description:Dry pea (Pisum sativum) seeds are valuable sources of plant protein, dietary fiber, and starch, but their uses in food products are restricted to some extent due to several off-flavor compounds. Saponins are glycosylated triterpenoids and are a major source of bitter, astringent, and metallic off-flavors in pea products. β-amyrin synthase (BAS) is the entry point enzyme for saponin biosynthesis in pea and therefore is an ideal target for knock-out using CRISPR/Cas9 genome editing to produce saponin deficient pea varieties. Here, in an elite yellow pea cultivar (CDC Inca), LC/MS analysis identified embryo tissue, not seed coat, as the main location of saponin storage in pea seeds. Differential expression analysis determined that PsBAS1 was preferentially expressed in embryo tissue relative to seed coat and was selected for CRISPR/Cas9 genome editing. The efficiency of CRISPR/Cas9 genome editing of PsBAS1 was systematically optimized in pea hairy roots. From these optimization procedures, the AtU6-26 promoter was found to be superior to the CaMV35S promoter for gRNA expression, and the use of 37°C was determined to increase the efficiency of CRISPR/Cas9 genome editing. These promoter and culture conditions were then applied to stable transformations. As a result, a bi-allelic mutation (deletion and inversion mutations) was generated in the PsBAS1 coding sequence in a T1 plant, and the segregated psbas1 plants from the T2 population showed a 99.8% reduction of saponins in their seeds. Interestingly, a small but statistically significant increase (~12%) in protein content with a slight decrease (~5%) in starch content was observed in the psbas1 mutants under phytotron growth conditions. This work demonstrated that flavor-improved traits can be readily introduced in any pea cultivar of interest using CRISPR/Cas9. Further field trials and sensory tests for improved flavor are necessary to assess the practical implications of the saponin-free pea seeds in food applications.

Dataset Information

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

Publications

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

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