Field trials and baking studies of ultra-low asparagine, genome edited (CRISPR/Cas9) and mutant (TILLING) wheat

Field trials were conducted of wheat (Triticum aestivum) cv. Cadenza in which asparagine synthetase gene, TaASN2, had been knocked out, either on its own or together with a partial knockout of the related gene, TaASN1, using CRISPR/Cas9. Chemical mutagenesis (TILLING) TaASN2 nulls in the Claire background were also included. The main aim was to assess the free asparagine content of the grain and the conversion of free asparagine to acrylamide, a toxic contaminant, in bread, toast and biscuits. Over two years of trials combined, the TaASN2 and TaASN1/2 CRISPR knockouts resulted in a reduction of free asparagine in the grain of 59% and 93%, respectively, compared with Cadenza. The reduction in the TaASN2 total knockout TILLING line compared with Claire was 50%. Yield was not affected in the edited lines but was reduced in the TILLING lines. Acrylamide in bread made from a TaASN1/2 CRISPR line was below detection levels, while in a TaASN2 CRISPR line it was 14% of the Cadenza control. Even after four minutes of toasting, acrylamide levels remained at 8% and 23%, respectively, of the control. The concentration in bread made from the TILLING TaASN2 knockout was 21% that for the Claire control, rising to 46% after four minutes of toasting. Acrylamide in biscuits made from a TaASN1/2 CRISPR line was reduced by 93% compared with the control. The relationship between acrylamide and colour was altered in the edited and mutant lines compared with the controls, with less acrylamide forming for the same degree of colour.