We reported in this journal in 2021 the generation of wheat genotypes in which the asparagine synthetase gene, TaASN2, had been ‘knocked out’ using CRISPR-Cas9 (Raffan et al., 2021). The editing had been achieved by introducing genes encoding the Cas9 nuclease, four guide RNAs (gRNAs) and a Bar marker gene into wheat (Triticum aestivum) cv. Cadenza. Here we report the results of a field trial of Line 178.35, an A genome null for TaASN2, and total nulls, 23.60 and 23.75 (Raffan et al., 2021). Also included were four AB genome nulls, referred to as TILLING lines 1–4, derived from a selected line of a mutant population produced by ethyl methanesulphonate treatment of wheat cv. Cadenza seeds (Rakszegi et al., 2010). The mutated TaASN2-A2 gene from this line was backcrossed into the cv. Claire background to generate AB genome nulls (cv. Claire lacks a B genome TaASN2 gene due to a ‘natural’ deletion (Oddy et al., 2021)).

The gRNA and Bar genes were segregating in all three GE lines, whilst only line 23.60 still contained the Cas9 gene. Consent to release the GMOs was granted by the UK government on 31/08/2021 (reference 21/R08/01). To our knowledge, the field trial was the first in Europe to include genome-edited (GE) wheat lines. A second total null GE line that had been produced, Line 59 (Raffan et al., 2021), was found to have additional edits in the closely related gene, TaASN1. Because these additional edits were not described in the application for consent to release the GMOs, this line was not included in the field trial.

The aim of knocking out TaASN2 was to reduce the concentration of free asparagine in the grain. Free asparagine is converted to the carcinogenic processing contaminant, acrylamide, during high-temperature processing, baking and toasting (Raffan and Halford, 2019). The concentrations of free asparagine in the grain of line 178 plants grown under glass were 56% and 68% of controls over two generations, while those of line 23 were 43% and 57% of controls. The aim of the field trial was to determine whether the low asparagine phenotype was maintained under field conditions and assess the performance of the lines with respect to emergence, yield, thousand grain weight (TGW) and composition.

The field trial was sown at the Rothamsted farm, Harpenden, UK, on 26/10/2021. It comprised 56 plots of 6 × 1.8 m (Figure 1a), with five plots of each of GE lines 178.35, 23.60 and 23.75, TILLING lines 1–4, a TILLING control, which had come through the backcross process but contained no mutations in TaASN2, plus 8 plots each of Cadenza and Claire. A test prior to drilling showed a germination rate of >95% for all of the lines, and all plots showed emergence by 01/12/2021.

我们在 2021 年的该期刊中报道了小麦基因型的产生，其中天冬酰胺合成酶基因TaASN2已使用 CRISPR-Cas9 被“敲除”（Raffan等人，  2021 年）。编辑是通过将编码 Cas9 核酸酶、四个引导 RNA (gRNA) 和一个Bar标记基因的基因引入小麦 ( Triticum aestivum ) cv中实现的。华彩乐段。在这里，我们报告了 178.35 系的田间试验结果， TaASN2的 A 基因组为空，总空值为 23.60 和 23.75（Raffan等人，  2021 年）). 还包括四个 AB 基因组空值，称为 TILLING 系 1-4，源自通过甲磺酸乙酯处理小麦品种产生的突变种群的选定系。Cadenza 种子（Rakszegi等人，  2010 年）。来自该品系的突变的TaASN2-A2基因被回交到 cv. 产生 AB 基因组空值的 Claire 背景（由于“自然”缺失， cv. Claire 缺少 B 基因组TaASN2基因（Oddy等人，  2021 年））。

gRNA 和Bar基因在所有三个 GE 品系中分离，而只有品系 23.60 仍包含Cas9基因。英国政府于 2021 年 8 月 31 日批准发布转基因生物（参考文献 21/R08/01）。据我们所知，该田间试验是欧洲首次包括基因组编辑 (GE) 小麦品系。已生产的第二个完全无效的 GE 品系，品系 59（Raffan等人，  2021 年）被发现在密切相关的基因TaASN1中具有额外的编辑。因为这些额外的编辑没有在批准释放转基因生物的申请中描述，所以该品系不包括在田间试验中。

敲除TaASN2的目的是降低谷物中游离天冬酰胺的浓度。在高温加工、烘烤和烘烤过程中，游离天冬酰胺会转化为致癌加工污染物丙烯酰胺（Raffan 和 Halford，  2019 年）。玻璃下栽培的178号系两代植物籽粒中游离天冬酰胺浓度分别为对照的56%和68%，而23号系为对照的43%和57%。田间试验的目的是确定低天冬酰胺表型是否在田间条件下得以维持，并评估品系在出苗率、产量、千粒重 (TGW) 和成分方面的表现。

田间试验于 2021 年 10 月 26 日在英国哈彭登的 Rothamsted 农场播种。它包括 56 个 6 × 1.8 m 的地块（图 1a），每个 GE 品系 178.35、23.60 和 23.75、TILLING 品系 1-4、TILLING 对照有五个地块，它通过回交过程但在TaASN2，加上 Cadenza 和 Claire 各 8 个地块。钻孔前的测试显示所有品系的发芽率 > 95%，并且所有地块都显示在 2021 年 1 月 12 日出现。