Sequencing of genome-edited wheat lines derived from cultivar Fielder

Wheat immunotoxicity is associated with abnormal reaction to gluten-derived peptides. Attempts to reduce immunotoxicity using breeding and biotechnology often affect dough quality. Here, the multiplexed CRISPR-Cas9 editing of cultivar Fielder was used to modify gluten-encoding genes, specifically focusing on omega- and gamma-gliadin gene copies, which were identified to be abundant in immunoreactive peptides based on the analysis of wheat genomes assembled using the long-read sequencing technologies. The whole genome sequencing of an edited line showed mutation or deletion of nearly all omega-gliadin and half of the gamma-gliadin gene copies and confirmed the lack of editing in the alpha/beta-gliadin genes. The estimated 75% and 64% reduction in omega- and gamma-gliadin content, respectively, had no negative impact on the end-use quality characteristics of grain protein and dough. A 47-fold immunoreactivity reduction compared to non-edited line was demonstrated using antibodies against immunotoxic peptides. Our results indicate that the targeted CRISPR-based modification of the omega- and gamma-gliadin gene copies determined to be abundant in immunoreactive peptides by analyzing high-quality genome assemblies is an effective mean for reducing immunotoxicity of wheat cultivars while minimizing the impact of editing on protein quality.

小麦的免疫毒性与其对谷蛋白衍生物肽的异常反应密切相关。采用育种和生物技术手段降低免疫毒性时，往往会对面团质量产生影响。本研究中，通过采用多路复用CRISPR-Cas9编辑技术对Fielder品种进行了基因编辑，旨在修改编码谷蛋白的基因，特别是针对omega-和gamma-谷蛋白基因拷贝，这些基因拷贝在利用长读长测序技术组装的小麦基因组分析中被发现富集于免疫反应性肽段中。对编辑后的品种进行的全基因组测序显示，几乎所有omega-谷蛋白基因拷贝以及一半的gamma-谷蛋白基因拷贝发生了突变或缺失，同时证实了alpha/beta-谷蛋白基因未受到编辑。omega-和gamma-谷蛋白含量的估计分别降低了75%和64%，这对谷物蛋白和面团的使用品质特征没有产生负面影响。通过针对免疫毒性肽段的抗体进行测试，与非编辑线相比，免疫反应性降低了47倍。我们的结果表明，通过对分析高质量基因组组装中富集于免疫反应性肽段的omega-和gamma-谷蛋白基因拷贝进行靶向CRISPR修改，是降低小麦品种免疫毒性的有效方法，同时最大限度地减少了编辑对蛋白质质量的影响。