Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula
Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula
复制标题
CRISPR/Cas9 系统在豆科植物蒺藜苜蓿模型中的靶向诱变
DOI：
10.1007/s00299-016-2069-9
复制
发表时间：
2017-02-01
影响因子：
6.2
通讯作者：
Lin, Hao
中科院分区：
文献类型：
--
作者：
Meng, Yingying;Hou, Yaling;Lin, Hao
近年来，已经开发了几种基于工程核酸酶的基因组编辑技术，并成功报道了各种生物体中的特定基因座的突变。与早期开发的基因组编辑方法相比，如锌指核酸酶（ZFN）和转录激活因子样效应物核酸酶（TALEN），它们需要对每个DNA靶标进行大量的蛋白质工程改造，成簇的规则散布的短回文重复序列（CRISPR）/Cas9系统仅需要改变单向导RNA（sgRNA）内的20-nt靶向序列。克隆策略的简单性和对潜在靶位点的较少限制的优点使得CRISPR/Cas9系统成为靶向基因组编辑的突出选择，以理解基因功能并在模型植物和粮食作物中开发有价值的性状（Li et al. 2013; Shan et al. 2013）。苜蓿（Medicago sativa）是多年生豆科植物，是世界上最重要的饲料作物之一。苜蓿因其高饲用价值和高产量而被公认为“饲料作物皇后”，但苜蓿同源四倍体基因组复杂，基因组学工具有限，阻碍了苜蓿的遗传改良和改良。蒺藜苜蓿是苜蓿的近缘种，具有二倍体遗传、基因组小、生命周期短、自然多样性高等特点。随着其基因组测序的完成，M.蒺藜草已被用作豆科牧草遗传学和基因组学研究的模式。最近，有报道称CRISPR/Cas9系统可以使M.通过根毛转化对蒺藜和大豆进行了研究，表明CRISPR/Cas9在豆类中的应用潜力（Michno等人，2015）。然而，在豆科牧草中基于CRISPR/Cas9的稳定转化尚未报道。在这项研究中，我们开发了一种高效的CRISPR/Cas9系统，通过农杆菌介导的转化，在模式豆科植物M中进行靶向基因突变。这将有助于苜蓿基因功能的研究，也扩大了豆科牧草基因组学的工具箱。为了提高向导RNA在M.在truncatula中，我们首先克隆了天然苜蓿U6启动子以驱动特异性sgRNA的表达（补充图10）。S1、S2）的情况下，为了使苜蓿CRISPR/Cas9系统适应农杆菌介导的转化，我们基于pFGC 5941的骨架构建了用于共表达指导RNA和Cas9的双元载体pFGC 5941-Cas9。所设计的二元载体含有由CaMV 35 S启动子驱动的密码子优化的化脓性链球菌Cas9基因（Shan et al.
In recent years, several genome-editing technologies based on engineered nucleases have been developed and successfully reported to mutate specific loci in various organisms. Compared to the early developed genome-editing methods, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which need substantial protein engineering to each DNA target, the clustered regularly interspersed short palin-dromic repeats (CRISPR)/Cas9 system only requires a change in a 20-nt targeting sequence within the single-guide RNA (sgRNA). The advantage of the simplicity of the cloning strategy and less limitations on potential target sites make the CRISPR/Cas9 system the standout choice for targeted genome editing in understanding gene function and developing valuable traits in model plants and food crops (Li et al. 2013; Shan et al. 2013). Alfalfa (Medicago sativa) is a perennial legume cultivated as one of the most important forage crops in the world. Alfalfa is well recognized as ''The queen of forage crops'' because of its high feed value and high yield productivity , yet the complex autotetraploid genome and limited genomics tools hamper the genetic modifications and improvements in alfalfa. Medicago truncatula is a close relative of alfalfa with a diploid genetics, small genomes, short lifecycle, and highly natural diversity. With the completion of its genome sequencing, M. truncatula has been adopted as a model for the study of genetics and genomics for forage legume. Recently, the CRISPR/Cas9 system has been reported to mutate targeted genes in somatic cells of M. truncatula and soybean by root hair transformation suggesting the application potential of CRISPR/Cas9 in legumes (Michno et al. 2015). However, CRISPR/Cas9-based stable transformation in forage legumes has not been reported. In this study, we developed an efficient CRISPR/Cas9 system through agrobacterium-mediated transformation for targeted gene mutations in the model legume M. truncatula, which will facilitate investigation in Medicago gene function and also expand the forage legume's genomics tool box. To promote the expression efficiency of guide RNA in M. truncatula, we first cloned a native Medicago U6 promoter to drive the expression of specific sgRNA (Supplementary Figs. S1, S2). To accommodate the Medicago CRISPR/Cas9 system to Agrobacterium-mediated transformation , we constructed a binary vector pFGC5941-Cas9 based on the backbone of pFGC5941 for co-expression of the guide RNA and Cas9. The designed binary vector contains a codon-optimized streptococcus pyogenes Cas9 gene driven by the 2 9 CaMV 35S promoters (Shan et al.