Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter
Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter
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DOI：
10.1007/s00299-017-2202-4
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发表时间：
2017-12-01
影响因子：
6.2
通讯作者：
Jacob, Yannick
中科院分区：
文献类型：
--
作者：
Zhang, Fei;LeBlanc, Chantal;Jacob, Yannick
柑橘是世界上最重要的水果作物之一。2015年，全球商业橙子（Citrus sinensis）产量超过4700万吨（mt）（https：//apps. fas。美国农业部。gov/psdonline/circulars/citrus. pdf）和各种其他柑橘栽培品种（普通话/柑橘，28公吨;葡萄柚，6公吨;酸橙/柠檬，7公吨）也是柑橘产业的重要商业产品。大多数柑橘属物种的世代时间为6年或更长，这使得使用传统育种方法产生具有所需性状的新品种既耗时又费力。此外，柑橘属植物的高度杂合性和广泛的无融合生殖阻碍了所需性状通过回交的整合。所有这些特点使得柑橘的遗传分析和基因功能研究具有挑战性。先前在柑橘中使用比较转录组分析或全基因组关联研究的工作表明了许多基因在发育、生物或非生物胁迫反应中的可能作用，但这些基因中很少有功能性特征（Wang et al. 2017; Wu et al. 2014; Xu et al. 2013）。因此，需要新的工具来快速产生柑橘突变，用于基础研究和作物改良。新的基因组工程技术为寻求在柑橘基因组中创造突变的研究人员提供了一条有希望的途径。例如，最近开发的CRISPR/Cas9（成簇规则间隔短回文重复序列/CRISPR相关Cas9）基因组编辑工具已成功用于许多植物物种，包括拟南芥、烟草、短柄草、水稻、玉米和小麦（Ma等人的综述2016）。然而，一些研究表明，与其他物种相比，柑橘中CRISPR/Cas9系统的编辑效率相当低（Jia和Wang 2014; Jia，et al. 2017; Peng，et al. 2017）。为了克服这一限制，我们开发了一种高效的CRISPR系统，该系统依赖于：（1）由拟南芥YAO启动子驱动的Cas9和（2）用于鉴定具有高表达Cas9的转基因柑橘植物的双功能选择标记。我们通过在柑橘中进行PDS（八氢番茄红素去饱和酶）基因的靶向诱变来证明该系统的效率。拟南芥YAO启动子已显示增加A.与由花椰菜花叶病毒（CaMV）35 S启动子驱动的Cas9相比，在拟南芥中的表达（Yan等人，2015）。由于柑橘属和拟南芥属是锦葵目中密切相关的属，并且YAO同源物存在于几种柑橘属物种的基因组中（图S1），我们推断在柑橘属中在拟南芥YAO启动子下表达Cas9也可以导致靶向突变的高效率。为了测试这一点，我们修饰了pYAO：hSpCas 9二元载体（Yan等人，2015）以用于柑橘。我们首先替换pYAO：hSpCas 9中的HPT基因（潮霉素磷酸转移酶），
Citrus is one of the most important fruit crops in the world. Global commercial orange (Citrus sinensis) production exceeded 47 million tons (mt) in 2015 (https://apps. fas. usda. gov/psdonline/circulars/citrus. pdf), and various other Citrus cultivars (mandarin/tangerine, 28 mt; grapefruit, 6 mt; lime/lemon, 7 mt) are also important commercial products for the Citrus industry. Most Citrus species have generation times of 6 years or more, making it time-consuming and laborious to generate new varieties with desired traits using traditional breeding approaches. In addition, high levels of heterozygosity and widespread apomixis in Citrus hinder the integration of desired traits through backcrossing. All these features make it challenging to carry out genetic analyses and gene function studies in Citrus. Previous work in Citrus using comparative transcriptome analyses or genome-wide association studies have indicated possible roles for a number of genes in development, biotic or abiotic stress responses, but few of these genes have been functionally characterized (Wang et al. 2017; Wu et al. 2014; Xu et al. 2013). Thus, new tools are needed to rapidly generate mutations in Citrus for basic research and crop improvement. New genome engineering technologies offer a promising route for researchers seeking to create mutations in the Citrus genome. For example, the recently developed CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated Cas9) genomeediting tool has been successfully used in many plant species, including Arabidopsis, tobacco, Brachypodium, rice, maize and wheat (reviewed in Ma, et al. 2016). However, several studies have shown that the editing efficiency of the CRISPR/Cas9 system in Citrus is quite low compared to these other species (Jia and Wang 2014; Jia, et al. 2017; Peng, et al. 2017). To overcome this limitation, we developed a highly efficient CRISPR system that relies on:(1) Cas9 driven by the Arabidopsis YAO promoter and (2) a bifunctional selectable marker used to identify transgenic Citrus plants with high expression of Cas9. We demonstrate the efficiency of this system by performing targeted mutagenesis of the PDS (phytoene desaturase) gene in Citrus.Expression of Cas9 under the A. thaliana YAO promoter has been shown to increase the amount of targeted mutations at a specific locus in A. thaliana as compared to Cas9 driven by the Cauliflower mosaic virus (CaMV) 35S promoter (Yan et al. 2015). Because Citrus and Arabidopsis are closely related genera within the order Malvidae, and a YAO homolog is present in the genomes of several Citrus species (Fig. S1), we reasoned that expression of Cas9 under the Arabidopsis YAO promoter in Citrus could also result in high efficiency of targeted mutations. To test this, we modified the pYAO: hSpCas9 binary vector (Yan et al. 2015) for use in Citrus. We first replaced the HPT gene (hygromycin phosphotransferase) in the pYAO: hSpCas9