Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Abstract

Summary We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and 1H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.

Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System

“…In addition, the combination of two sgRNAs on one target gene used in our study, could explain the increase in the efficiency obtained. Such strategy not only increased the possibilities of inducing mutations in the target gene, but also led to the elimination of larger, specific fragments from the coding sequence as was previously reported in tomato (Brooks et al, 2014), rice (Zhou et al, 2014), barley (Kapusi et al, 2017) and potato (Tuncel et al, 2019;Veillet et al, 2019).…”

Production of very‐high‐amylose cassava by post‐transcriptional silencing of branching enzyme genes

“…Silencing the expression of BE isoforms in potato showed increased tuber yield but with reduced starch content (Hofvander et al 2004). Cas9‐mediated mutagenesis of SBE1 and SBE2 in tetraploid potatoes was recently reported to generate tuber starches with a range of distinct properties (Tuncel et al 2019). Similarly, reduced BE2 expression in sweet potato led to increased amylose content and reduced starch yield (Shimada et al 2006).…”

“…Enzymatic activity of BEs is regulated by phosphorylation and protein–protein interactions (Tetlow et al 2004). Suppression or mutation of BE function results in the increase of amylose content in many plants, for example, potato (Schwall et al 2000; Tuncel et al 2019), wheat (Sestili et al 2010), rice (Sun et al 2017), and sweet potato (Shimada et al 2006). Normally, high‐amylose starches show altered properties in applications distinguishable from the native or waxy starches, including their delayed gelatinization temperature, high gelling capacity, and easy forming film (Richardson et al 2000; Zhou et al 2015; Wang et al 2017).…”

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

“…Only a few protocols for genome editing and protoplast regeneration have been developed, all of which were achieved in dicots (Andersson et al 2017(Andersson et al , 2018Hsu et al 2019;Jin et al 2019;Lin et al 2018;Tuncel et al 2019;Woo et al 2015). Many protoplast regeneration protocols are available in other species, including rice (Shimamoto et al 1989;Toriyama et al 1988) and other important Poaceae species (Rhodes et al 1988a(Rhodes et al , 1988b.…”