An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.
An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.
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玉米中高频靶向诱变的农杆菌销售的CRISPR/CAS9系统。
DOI：
10.1111/pbi.12611
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发表时间：
2017-02
影响因子：
13.8
通讯作者：
Yang B
中科院分区：
文献类型：
--
作者：
Char SN;Neelakandan AK;Nahampun H;Frame B;Main M;Spalding MH;Becraft PW;Meyers BC;Walbot V;Wang K;Yang B
CRISPR/Cas9是许多生物体中强大的基因组编辑工具，包括许多单子叶和双子叶植物。虽然与其他基于核酸酶的基因组编辑工具相比，CRISPR/CAS9的设计和应用更简单，但优化需要考虑特定物种的DNA传递和组织再生方法，以实现准确性和效率。在这里，我们描述了一个公共部门系统，ISU玉米CRISPR，利用农杆菌传递的CRISPR/Cas9在玉米中进行高频定向突变。该系统由大肠杆菌克隆载体和农杆菌双元载体组成。它可以用来克隆最多四个引导RNA，用于单基因或多基因靶向。我们利用两对重复的四个玉米基因：ArgAerte18(ZmAgo18a和ZmAgo18b)和二氢黄酮醇4-还原酶或无花青素基因(A1和A4)对该系统的诱变频率和遗传力进行了评估。在Hi-II和B104两种基因型中，携带一个基因座的单等位基因突变或双等位基因突变以及两个基因座的各种等位基因突变组合的T0转基因事件的突变率均超过70%。通过遗传分离，在T1代中可以产生只携带所需突变等位基因而不携带CRISPR转基因的零分离株。一个活跃的CRISPR/Cas9转基因遗传导致在后续世代中发生额外的靶标特异性突变。通过混合含有不同Cas9/gRNA模块的两个单独的农杆菌菌株对未成熟胚胎进行双重感染可以提高成本效益。综上所述，这些发现表明ISU玉米CRISPR平台是一个有效和强大的工具，在玉米中进行定向突变。
CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.