Camelina (Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.

中文翻译：

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通过 CRISPR 介导的透明种皮 8 破坏，创造出具有增强油脂积累的黄色种子亚麻荠 (Camelina sativa)


亚麻荠 ( Camelina sativa L.) 是十字花科的六倍体成员，是一种新兴的油料作物，其开发是为了满足作为生物燃料原料的植物油日益增长的需求。在其他芸苔属植物中，高含油量可能与黄色种子表型有关，而这对于亚麻荠来说是未知的。我们试图使用 CRISPR/Cas9 技术来破坏其透明种皮 8 (TT8) 转录因子基因并评估由此产生的种子表型，从而创造黄籽亚麻荠。我们鉴定了三个TT8基因，三个亚麻荠亚基因组中各有一个，并获得了包含移码编辑的独立CsTT8系。 TT8的破坏导致种皮颜色从棕色变为黄色，反映出它们的黄酮类积累量减少了高达 44%，并且组织良好的种皮粘液层丧失。 CsTT8编辑种子的转录组分析显示，脂质相关转录因子LEC1 、 LEC2 、 FUS3和WRI1及其下游脂肪酸合成相关靶标的表达显着增加。这些变化导致代谢重塑，脂肪酸合成率增加，总脂肪酸 (TFA) 积累相应增加，从种子重量的 32.4% 增加到高达 38.0%，TAG 产量增加 21% 以上，淀粉或蛋白质没有显着变化与亲本系相比的水平。这些数据凸显了 CRISPR 在亚麻荠中创造新型强化油种质的有效性。由此产生的品系可能会直接有助于未来的净零碳能源生产，或者与其他特性相结合，以高产量生产所需的脂质衍生生物产品。