Nature Plants ( IF 18.0 ) Pub Date : 2021-11-15 , DOI: 10.1038/s41477-021-01019-4 Yu Lu 1 , Jiyao Wang 2 , Bo Chen 2 , Sudong Mo 2 , Lei Lian 2, 3 , Yanmin Luo 2 , Dehui Ding 2 , Yanhua Ding 2 , Qing Cao 2 , Yucai Li 2 , Yong Li 2 , Guizhi Liu 2 , Qiqi Hou 2 , Tingting Cheng 2 , Junting Wei 2 , Yanrong Zhang 2 , Guangwu Chen 2 , Chao Song 2 , Qiang Hu 2 , Shuai Sun 4, 5 , Guangyi Fan 5 , Yating Wang 1 , Zhiting Liu 1 , Baoan Song 3 , Jian-Kang Zhu 6 , Huarong Li 2 , Linjian Jiang 1
Structural variations (SVs), such as inversion and duplication, contribute to important agronomic traits in crops1. Pan-genome studies revealed that SVs were a crucial and ubiquitous force driving genetic diversification2,3,4. Although genome editing can effectively create SVs in plants and animals5,6,7,8, the potential of designed SVs in breeding has been overlooked. Here, we show that new genes and traits can be created in rice by designed large-scale genomic inversion or duplication using CRISPR/Cas9. A 911 kb inversion on chromosome 1 resulted in a designed promoter swap between CP12 and PPO1, and a 338 kb duplication between HPPD and Ubiquitin2 on chromosome 2 created a novel gene cassette at the joint, promoterUbiquitin2::HPPD. Since the original CP12 and Ubiquitin2 genes were highly expressed in leaves, the expression of PPO1 and HPPD in edited plants with homozygous SV alleles was increased by tens of folds and conferred sufficient herbicide resistance in field trials without adverse effects on other important agronomic traits. CRISPR/Cas-based genome editing for gene knock-ups has been generally considered very difficult without inserting donor DNA as regulatory elements. Our study challenges this notion by providing a donor-DNA-free strategy, thus greatly expanding the utility of CRISPR/Cas in plant and animal improvements.
中文翻译:
一种无供体 DNA 的基于 CRISPR/Cas 的水稻基因敲除方法
结构变异 (SV),如倒置和重复,有助于作物1的重要农艺性状。泛基因组研究表明,SV 是推动基因多样化的关键且无处不在的力量2,3,4。尽管基因组编辑可以有效地在植物和动物中创造 SVs 5,6,7,8,但设计的 SVs 在育种中的潜力却被忽视了。在这里,我们展示了通过使用 CRISPR/Cas9 设计的大规模基因组倒置或复制,可以在水稻中创造新的基因和性状。1 号染色体上的 911 kb 倒位导致CP12和PPO1之间设计的启动子交换,以及HPPD和泛素2之间的 338 kb 重复在 2 号染色体上,在关节处创建了一个新的基因盒,启动子Ubiquitin2 ::HPPD。由于原始CP12和Ubiquitin2基因在叶片中高表达,因此PPO1和HPPD的表达在具有纯合 SV 等位基因的经过编辑的植物中增加了数十倍,并在田间试验中赋予了足够的除草剂抗性,而对其他重要的农艺性状没有不利影响。在不插入供体 DNA 作为调控元件的情况下,基于 CRISPR/Cas 的基因组编辑通常被认为非常困难。我们的研究通过提供无供体 DNA 的策略来挑战这一概念,从而极大地扩展了 CRISPR/Cas 在植物和动物改良中的效用。