Plant Sciences and the Bioeconomy, Rothamsted Research, Harpenden, UK.
National Agri-Food Biotechnology Institute (NABI), Mohali, India.
FEBS Lett. 2022 Aug;596(15):1865-1870. doi: 10.1002/1873-3468.14365. Epub 2022 May 6.
Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual-guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis-related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase-deficient sugar-dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20-fold and the triacylglycerol content increases by around 30-fold. This deletion strategy offers a transgene-free route to engineering traits that rely on transcriptional gain-of-function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.
在不插入外源 DNA 的情况下获得功能获得表型是植物生物技术学家面临的一个重要挑战。在这里，我们展示了一种通过使用双向导 CRISPR/Cas9 系统删除基因间基因组序列，从而实现基因在启动子控制下表达的方法。我们将一个非必需的光合作用相关基因的启动子与拟南芥脂酶缺陷型糖依赖 1 突变体中的 DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) 融合，以驱动叶片中外源油脂的积累。DGAT2 的表达增强了 20 多倍，三酰基甘油的含量增加了约 30 倍。这种删除策略为工程性状提供了一种无转基因的途径，这些性状依赖于转录功能获得，例如生产高脂质饲料以提高反刍动物养殖的生产力和可持续性。
