背景:大豆是世界范围内种植的作物,由于其在食品中的应用,饲料,和生物柴油工业。大豆的基因组编辑始于ZFN和TALEN技术;然而,CRISPR/Cas已经出现,并很快成为大豆基因组操作的首选方法,因为它更精确,易于处理,和成本效益。最近的报道集中在常规的Cas9核酸酶上,Cas9切口酶(nCas9)衍生的基础编辑器,Cas12a(正式为Cpf1)是大豆中最常用的基因组编辑。尽管如此,为了有效编辑优良大豆品种的基因组,需要克服复杂植物基因工程流程中的几个挑战。这些挑战包括(1)优化CRISPR盒设计(即,gRNA和Cas启动子,gRNA设计和测试,gRNA的数量,和二进制向量),(2)提高变换频率,(3)提高靶向植物细胞的编辑效率,(4)提高大豆作物产量。
目的:这篇综述概述了使用CRISPR/Cas技术进行大豆基因组编辑,讨论当前的挑战,并强调克服现有瓶颈的理论(见解)和实践建议。
■CRISPR/Cas系统是细菌先天免疫系统的一部分。它已被用作基因组编辑的生物技术工具,并有效地应用于大豆以揭示基因功能,改善产量和营养籽粒品质等农艺性状,增强生物和非生物胁迫耐受性。迄今为止,编辑效率已通过原生质体和毛状根测定得到验证,而稳定的植物转化依赖于农杆菌介导的和粒子轰击方法。然而,CRISPR/Cas工作流程的大多数步骤都需要优化才能在大豆植物中实现更有效的基因组编辑。
BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production.
OBJECTIVE: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks.
UNASSIGNED: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the efficiency of gRNAs has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.