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

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. 2019), the European Commission issued Recommendation (EU) 2019/1888, which stated that levels of acrylamide should be monitored in a range of foods that did not fall within the scope of Regulation (EU) 2017/2158, including a number of bakery and cereal products (European Commission, 2019). Methods for reducing acrylamide in food products have been compiled in an 'Acrylamide Toolbox' by FoodDrinkEurope (2019). Some have been successful, but they are not applicable to all food types, are often expensive to implement and may have detrimental effects on product quality. The food industry would therefore benefit from the availability of raw materials with lower 1602

Current Status and Perspectives on the Application of CRISPR/Cas9 Gene-Editing System to Develop a Low-Gluten, Non-Transgenic Wheat Variety

“…Cadenza using the CRISPR/Cas9 system. The study provided strong evidence that very low-asparagine commercial wheat varieties can be produced, allowing for the development of lower-acrylamide bread, cereals, biscuits, and other wheat-based foods [70].…”

“…It was also demonstrated that having only one wild-type copy of each of the three TaNP1 genes was enough to maintain male fertility [69]. In a recent study, in order to reduce the expression of asparagine synthetase in grain without affecting its expression in any other part of the plant, Raffan et al (2021) targeted the TaASN2 gene in T. aestivum cv. Cadenza using the CRISPR/Cas9 system.…”

Crop Quality Improvement Through Genome Editing Strategy

“…Acrylamide in food is a processing contaminant that forms from free asparagine and potentially increases the risk of developing cancer for humans. In wheat, Raffan et al (2021) knocked out the asparagine synthetase gene TaASN2 using four guide RNAs targeting all three homologues of TaASN2. Compared with the wild type, the concentration of free asparagine in seeds of the plants with all six TaASN2 alleles edited was significantly decreased, up to 90%.…”

Can agroecology and CRISPR mix? The politics of complementarity and moving toward technology sovereignty

“…To take a more recent example, in May 2021, the UK's Rothamsted Research applied for a permit to field test wheat that has been gene-edited to contain less asparagine, an amino acid that becomes the carcinogen acrylamide when cooked at high temperatures (Rothamsted 2021;Raffan et al 2021). The research institute explained that the "low acrylamide" wheat was modified using CRISPR to reduce the production of this chemical, which may cause cancer.…”