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

Sarah Raffan, Caroline Sparks, Alison Hutley, Lucy S Hyde, Damiano Martignago, Andrew Mead, Steven J Hanley, Paul A Wilkinson, Gary L A Barker, Keith J Edwards, Tanya Curtis, Sarah L Usher, Andrej Kosik, Nigel G Halford

Research output: Contribution to journalArticle (Academic Journal)peer-review

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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.
Original languageEnglish
Pages (from-to)1602-1613
Number of pages12
JournalPlant Biotechnology Journal
Volume19
Issue number8
Early online date26 Feb 2021
DOIs
Publication statusPublished - Aug 2021

Bibliographical note

Funding Information:
SR was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) SWBio iCASE DTP Studentship, with partners: University of Bristol, Agriculture and Horticulture Development Board, KWS UK Ltd, Saaten Union UK Ltd, RAGT Seeds Ltd, Syngenta UK Ltd, and Limagrain UK Ltd. NH, CS and AH are supported at Rothamsted Research by the BBSRC the Designing Future Wheat Programme (BB/P016855/1). via

Funding Information:
SR was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) SWBio iCASE DTP Studentship, with partners: University of Bristol, Agriculture and Horticulture Development Board, KWS UK Ltd, Saaten Union UK Ltd, RAGT Seeds Ltd, Syngenta UK Ltd, and Limagrain UK Ltd. NH, CS and AH are supported at Rothamsted Research by the BBSRC via the Designing Future Wheat Programme (BB/P016855/1).

Publisher Copyright:
© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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