Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2

A - Papers appearing in refereed journals

Raffan, S., Sparks, C. A., Huttly, A. K., Hyde, L., Martignago, D., Mead, A., Hanley, S. J., Wilkinson, P., Barker, G., Edwards, K. J., Curtis, T. Y., Usher, S. L., Kosik, O. and Halford, N. G. 2021. Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2 . Plant Biotechnology Journal. 19 (8), pp. 1602-1613. https://doi.org/10.1111/pbi.13573

AuthorsRaffan, S., Sparks, C. A., Huttly, A. K., Hyde, L., Martignago, D., Mead, A., Hanley, S. J., Wilkinson, P., Barker, G., Edwards, K. J., Curtis, T. Y., Usher, S. L., Kosik, O. and Halford, N. G.
Abstract

Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high-temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4-gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.

KeywordsAsparagine synthetase; Acrylamide; Food safety; Wheat; Grain composition; Genome editing; CRISPR/Cas9; Amino acids
Year of Publication2021
JournalPlant Biotechnology Journal
Journal citation19 (8), pp. 1602-1613
Digital Object Identifier (DOI)https://doi.org/10.1111/pbi.13573
Open accessPublished as ‘gold’ (paid) open access
FunderBiotechnology and Biological Sciences Research Council
Funder project or codeSWBio DTP PhD studentship
DFW - Designing Future Wheat - Work package 1 (WP1) - Increased efficiency and sustainability
Publisher's version
Accepted author manuscript
Supplemental file
Supplemental file
Output statusPublished
Publication dates
Online26 Feb 2021
PublisherWiley
ISSN1467-7644

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