Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato

Abstract

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.

Starch branching enzymes as putative determinants of postharvest quality in horticultural crops

“…This can be achieved by either physical, chemical, or enzymatic modifications of purified starch, e.g., etherification, esterification, or by fine-tuning the activity of starch biosynthetic enzymes [ 129 , 130 ]. Reduction or knockout of SBEs in a range of species have reliably led to an increase in the resistant starch (RS) content in various species including horticultural crops e.g., potato, sweet potato, and cassava, [ 75 , 78 , 130 – 140 ]. Interestingly, SBE2 is not the dominant isoform expressed in storage tubers and roots, but it exerts a major function in amylopectin synthesis [ 141 ].…”

Potato trait development going fast-forward with genome editing

“…Mutations in the SBE2 gene alone gave a 15% decrease in the degree of branching, while tetra-allelic mutations in both SBE1 and SBE2 resulted in a more significant decrease, down to half that of wild-type starch. In a subsequent study, RNP transfection of protoplasts was used, and regenerated potato genotypes containing starch without any detectable branching and essentially free of amylopectin when all eight alleles had indels introduced [8]. In both studies, at least one allele in one or both of the genes was an in-frame mutation, and complete loss of both enzymes was speculated to detrimentally reduce vitality.…”

Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

“…In these crop varieties, CRISPR-mediated, DNA-free genome editing in protoplasts followed by regeneration into whole plants would be the most feasible way to directly apply gene editing technologies to improve traits and increase commercial value. This method has already been experimentally proven in protoplasts including potato (Andersson et al, 2017;Andersson et al, 2018;Tuncel et al, 2019;González et al, 2020;Zhao et al, 2021;Nicolia et al, 2021), N. tabacum (Lin et al, 2018;Hsu et al, 2019), N. benthamiana (Hsu et al, 2021a,b), Brassica oleracea (Park et al, 2020;Hsu C. T. et al, 2021), lettuce (Woo et al, 2015), petunia (Yu et al, 2020), and witloof (De Bruyn et al, 2020). The main steps of gene editing using protoplast regeneration are illustrated in Figure 1B.…”