Genome editing of five starch synthesis genes produces highly resistant starch and dietary fibre in barley grains

Resistant starch (RS) refers to starch that is not digested in the stomach or small intestine, and provides health benefits by reducing glycaemic index and promoting gut health (Hazard et al., 2020). Barley (Hordeum vulgare L.) is the fourth most widely cultivated cereal worldwide, and there is growing interest in barley as a healthy food. The RS content of cereal grains is positively associated with the presence of amylose and long-chain amylopectin (Li et al., 2021). However, increasing the levels of amylose in crops is still challenging. Overexpressing the enzyme involved in amylose synthesis, granule-bound starch synthase (GBSS), does not increase amylose content in most species (Seung, 2020). By contrast, suppressing amylopectin synthesis enzymes such as starch synthase (SS) and starch-branching enzyme (SBE) isoforms in cereals increases amylose content significantly (Chen et al., 2021). However, knockout mutations in these genes usually compromise the yield potential. Recent progress in CRISPR/Cas9-mediated gene editing make it possible to induce targeted mutations in multiplex genes (Cheng et al., 2023; Lawrenson et al., 2015; Luo et al., 2021), which provides a promising approach for devising new strategies to increase amylose content while avoiding/minimizing production limitations. We reasoned that multiplex editing could allow the contribution of all other SS and SBE isoforms to amylose content to be systematically assessed, and allow screening for optimum combinations of mutations that could synergistically provide strong increases in amylose content. Single-guide RNAs (sgRNAs) were designed to target the exons of seven desired genes encoding four SSs (SSI, SSIIa, SSIIIa and SSIV) and three SBEs (SBEI, SBEIIa and SBEIIb) based on the genome sequence of barley cv. Golden Promise (Table S1). The CRISPR/Cas9 vector was introduced into immature embryos of Golden Promise using Agrobacterium-mediated transformation (Method S1). We identified 113 edited plants from 152 T0 transformants, and these plants contained mutations in one to six of the targeted genes; however, no transgenic plants contained mutations in all seven target genes as no SSI mutant plants were identified (Figure 1a; Table S2). Sequencing showed that the nucleotide mutations in the target regions led to premature stop codons, which would disrupt the functional structures of the corresponding proteins (Figure 1b). In the T3 generation, we obtained 10 edited lines with different genotypes that were homozygous for mutations in one to three target genes. Western blotting of grain protein extracts from these lines showed that the target proteins were absent or dramatically reduced in abundance, consistent with the mutations disrupting normal protein accumulation (Figure 1c; Figure S1–S10). Notably in the ssIIa mutant, in addition to the absence of SSIIa, the abundance of SSI, SBEIIa and SBEIIb was also undetectable or strongly reduced relative to the wild type (WT). This is consistent with observations in ssIIa mutants of other cereal crops and can be explained by the formation of a multienzyme complex of SSI/SSIIa/SBEIIa or SBEIIb (Liu et al., 2012). Grains from the mutant lines exhibited varying degrees of shrunken morphology, and the SSIIa mutation induced the greatest negative effect on grain weight among the five monogenic mutants (Figure 1a). Interestingly, combining the SSIIa and SSIIIa mutations appeared to overcome this grain weight deficiency (Table S3). Scanning electron microscopy revealed that the starch granules from the edited lines exhibited a dramatically altered morphology with hollow, concave surface and sticky or amorphous shapes (Figure 1d). Significantly, the ssIIassIIIassIVa and ssIIasbeIIasbeIIb lines had a few sizable A-type granules that contained several small, concave granules with or without clear boundaries. The particle size distribution of starch granules showed that starches from the sbeIIa, sbeIIb and ssIIassIIIa mutants contained significantly more A-type granules but fewer B-type granules than the controls, while the other mutants had fewer A-type granules and more B-type granules than the controls (Figure 1e). The total starch contents of the 10 lines were significantly lower than that of the controls, and they all except ssIIassIVa and ssIIassIIIassIVa had significantly higher starch contents than the ssIIa mutant (Figure 1f; Table S4). Relative to the controls, apart from sbeIIb, which expressed a slight but not significant increase in the amylose content, the amylose contents of all mutants were greatly increased. Significantly, the ssIIasbeIIasbeIIb and sbeIIasbeIIb lines exhibited the highest amylose content (87.43% and 86.82%, respectively), which were around fourfold higher than control lines and 1.5-fold higher than the ssIIa mutant. Fluorescence-activated capillary electrophoresis indicated that inactivating the starch synthase genes also dramatically affected the chain-length distribution of amylopectin (Figure 1e). Furthermore, the thermal properties, swelling power and solubility of the starches in all mutants were striking changed compared with the controls (Figure 1g). Most of the edited lines contained greatly elevated contents of RS, β-glucan, fructan and fibre than the controls (Figure 1f; Table S4). In particular, the polygenic mutants sbeIIasbeIIb and ssIIasbeIIasbeIIb had extremely high RS (12.27% and 14.50%, respectively), which were 35-fold greater than that of the controls. The ssIIassIIIassIVa, ssIIasbeIIasbeIIb and ssIIassIVa mutants exhibited the highest fructan, β-glucan and fibre contents among all mutants, respectively; they were greater than that of the controls and the ssIIa. In summary, we successfully used multiplex editing to generate barley mutants harbouring single, double and triple mutations in five starch synthesis genes and produced either higher dietary fibre content or higher grain weights than those previously achieved by employing a single gene target (i.e., SSIIa). Three mutants, ssIIassIVa, sbeIIasbeIIb and ssIIasbeIIasbeIIb, exhibited improved levels of amylose and dietary fibre and/or a higher grain weight compared with the single ssIIa mutant. These were determined to be the best choices among the five polygenic mutants for future applications in the production of healthy food products. Our study demonstrates that Cas9-mediated multiplex gene editing is feasible for modifying starch to generate grain with higher RS contents than targeted editing of single genes and provides a theoretical basis and genetic resource for breeding barley with improved health benefits. This work was supported by the Sichuan Science and Technology Program, China (2023YFH0041). The authors declare no competing interests. QY conducted the experiments; JPR, ZYL, YMW, YLZ and QTJ prepared the manuscript; ZYL, YMW and QTJ conceptualized the project. The data that supports the findings of this study are available in the supplementary material of this article. Method S1 The protocol for barley transformation and starch property test. Figure S1 Western blot results of the targeted starch synthases in the ssIIa lines. Figure S2 Western blot results of the targeted starch synthases in the ssIIIa lines. Figure S3 Western blot results of the targeted starch synthases in the ssIV lines. Figure S4 Western blot results of the targeted starch synthases in the sbeIIa lines. Figure S5 Western blot results of the targeted starch synthases in the sbeIIb lines. Figure S6 Western blot results of the targeted starch synthases in the ssIIassIIIa lines. Figure S7 Western blot results of the targeted starch synthases in the ssIIassIVa lines. Figure S8 Western blot results of the targeted starch synthases in the sbeIIasbeIIb lines. Figure S9 Western blot results of the targeted starch synthases in the ssIIassIIIassIVa lines. Figure S10 Western blot results of the targeted starch synthases in the ssIIasbeIIasbeIIb lines. Table S1 sgRNAs and primers used in this study. Table S2 Genotypes of T0 transgenic barley plants. Table S3 Grain weights and sizes of mutant barley lines. Table S4 Starch compositions of mutant barley grains. Accessing Supplementary Material. 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