Genome Editing for Low-Acrylamide Wheat

Acrylamide (C3H5NO) is a food processing contaminant that has been classed as a probable (Group 2a) human carcinogen. Acrylamide forms from the reaction of free (non-protein) asparagine with reducing sugars during food processing. All major cereal products are affected and wheat products represent one of the main sources of dietary acrylamide intake in Europe. Asparagine concentration is the determining factor for acrylamide formation in cereal products. Asparagine biosynthesis is catalysed by a family of enzymes called asparagine synthetases (ASNs). The ASN genes were investigated and five ASN genes (TaASN1-4, with a double copy of TaASN3) identified in wheat (Triticum aestivum), with TaASN2 showing grain-specific expression. CRISPR/Cas9 was used to knock out the TaASN2 gene of wheat cv. Cadenza. A polycistronic gene containing four gRNAs, interspaced with tRNAs, was designed and introduced into wheat embryos by particle bombardment. The subsequent edits were characterised in the T1 and T2 generations using Next Generation Sequencing nucleotide sequence analysis. Triple (A, B, and D genome) nulls were identified, alongside an AD and an A genome null. Amino acid concentrations were measured in the T2 and T3 seed, with one triple null line showing a substantial reduction in the free asparagine concentration in the grain (90 % in the T2 seed and 50 % in the T3 seed compared with wildtype). The free asparagine also reduced as a proportion of the total free amino acid pool. Significant effects were also seen in glutamate and aspartate concentrations. Free asparagine and total free amino acid concentrations were higher in the T3 than T2 seeds, probably due to heat stress, but the concentrations in the edited plants remained substantially lower than in wildtype. Some of the edited lines showed poor germination, but this could be overcome by application of exogenous asparagine and no other phenotype was noted