Multiplex CRISPR/Cas9-mediated raffinose synthase gene editing reduces raffinose family oligosaccharides in soybean

Abstract

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.

Keywords: CRISPR/Cas9; RFOs; RS; multiplex gene editing; soybean.

Figure 1

Schematic diagram of the five CRISPR/Cas9 expression systems. TCTU, two-component transcriptional unit system; STU, single transcriptional unit system; GmUBQ, Glycine max polyubiquitin promoter; NOS, nopaline synthase terminator; GmU6, soybean U6 promoter to express sgRNA; polyT, the terminator of GmU6; polyA, a synthetic polyA sequence to facilitate translation; Csy4, RNA endoribonuclease Csy4 (orange rectangle) from Pseudomonas aeruginosa; Csy4 cleavage site, a sequence recognized and cleaved by Csy4 (orange diamond); tRNA, 77-bp pre-tRNA^Gly gene; P2A, a ribosomal skipping peptide (gray rectangle); and LB/RB, the left and right borders of T-DNA.

Figure 2

A schematic presentation of the soybean genotyping results for all tested multiplex gene editing systems. Each column (including the four rectangles) represents one tested transgenic event; each rectangle indicates one sgRNA target of this event; the blue rectangles indicate target genes with editing; and the white rectangles indicate the wild type. Mutation efficiency was calculated as the number of mutants divided by the total number of tested events genotyped for each target site (efficiency = editing hairy roots/all hairy roots tested). “n” indicates the number of events tested for each editing system.

Figure 3

Soybean RS2 and RS3 gene structure and target sequence locations. (A) Target sequence of RS2 and RS3; the guide sequence is marked in red, and the PAM motif (NGG) is highlighted in green. The distance values between the two sgRNAs were 227 and 118 bp, respectively. (B) The schematic diagram of TCTU-tRNA-RS vector targeting RS2 and RS3.

Figure 4

Genotypes of the targeted regions in the RS2 and RS3 genes in soybean T0 transgenic plants. Targeted sequences (sgRNAs) are indicated by the red color, and the PAM sequences are highlighted in green. The distance values between the two sgRNAs were 227 and 118 bp, respectively. The base insertion is marked in purple; the base substitution is labeled in blue; and the base deletion is highlighted in yellow bars. “-” means deletion; “+” means insertion; and “sub1” means one base was substituted.

Figure 5

Inheritance and segregation of the RS2 and RS3 mutations in T1 transgenic plants. Targeted sequences (sgRNAs) are indicated by the red color, and the PAM sequences are highlighted in green. The distance values between the two sgRNAs were 227 and 118 bp, respectively. The base insertion is marked in purple; the base substitution is labeled in blue; and the base deletion is highlighted in yellow bars. “-” means deletion; “+” means insertion; and “sub1” means one base was substituted.

Figure 6

Selected homozygous double and single mutants of RS2 and RS3 in T2 transgenic plants. (A) Genotypes of selected RS2 and RS3 double and single mutants. Targeted sequences (sgRNAs) are indicated by the red color, and the PAM sequences are highlighted in green. The base insertion is marked in purple, and the base deletion is highlighted in yellow bars. “-” means deletion and “+” means insertion. (B) Sanger sequencing of RS2 and RS3 gene in double or single mutants. The arrow indicates the mutation location.

Figure 7

Growth and development phenotype of the RS2 and RS3 mutants. (A) Comparison of weight per 100 seeds in wild type and null mutants. The bar indicates 0.5 cm. (B) Pod length of the wild type and RS2 and RS3 null mutant lines. The bar indicates 1 cm. (C) Comparison of plant height in wild type and null mutants after 1-month of growth in the greenhouse. The bar indicates 5 cm. (D) Comparison of grain yield per single plant in wild type and null mutants. Means followed by the same letter were not significantly different from each other using the Student’s t-test analysis, P < 0.05.