In this review, the steps of development of the CRISPR/Cas9 genome editing system and its applications in various plant genomes were highlighted. The CRISPR/Cas9 genome editing technology originates from the prokaryotic adaptive immune systems that confer resistance to foreign genetic elements such as plasmids and phages. The natural CRISPR/Cas systems show extensive structural and functional diversity. Based on the Cas protein contents and amino acid sequences, the natural CRISPR/Cas systems have been classified into three major classes, Type I, TypeⅡ and Type III. The TypeⅡCRISPR/Cas system is the engineered one for targeted genome editing purpose in most of cases so far, as it needs optimization of the Cas expression and design of the sgRNA only. In 2013, the first applications of CRISPR/Cas9 genome editing technology in plants were published. Since then, the CRISPR/Cas9 system has been used in various plant species for targeted genome editing. Like ZFNs and TALENs, CRISPR/Cas9 system uses engineered nuclease to generate double-strand breaks (DSBs) on the targeted DNA site, and subsequently to stimulate cellular DNA repair mechanisms by exploiting either NHEJ pathway or HDR pathway to generate small insertions/deletions/genome modifications. CRISPR/Cas9 technology allows researchers to perform targeted mutagenesis on target genes of different crops, precisely and easily changing the sequences and functions of particular genes at exact chromosomal locations in different plant genomes. Compared with ZFNs and TALENs technologies, CRISPR/Cas9 genome editing system is based on RNA-guided engineered nucleases, and is easier to manipulate. Furthermore, CRISPR/Cas9 is capable of introducing DSBs at multiple sites. The potential of multiplexing provides practical advantages over ZFNs and TALENs technologies, to edit multiple target genes in the same pathway simultaneously. Due to the practical advantages of CRISPR/Cas9 over the other genome editing technologies, it establishes a prosperous outlook in gene discovery and trait development in crop genetic improvement and breeding studies. In this review, the possible applications of CRISPR/Cas9 genome editing technique in various aspects of plant genetics and breeding were also discussed, except the targeted genome editing. CRISPR/Cas9 genome editing technology is another stepping stone in utilizing genetic manipulation in genetic studies and breeding, after genetic modification. Unlike genetic modification, CRISPR/Cas9 genome editing technique generates phenotypic variation that is indistinguishable from that obtained through natural means or conventional mutagenesis. This ambiguity challenges the current GMO regulatory definitions, and provides a potential barrier for further use of CRISPR/Cas9 genome editing technique in crop genetics and breeding.%CRISPR/Cas9系统是近年发展起来的、由导向 RNA 介导的基因组定向编辑技术。总结了 CRISPR/Cas9基因组定向编辑技术的发展历程，并综述了其在作物遗传育种研究中的多方面应用。CRISPR/Cas系统是存在于大多数细菌与所有古生菌中的一种后天免疫系统,以消灭外来质体或者噬菌体。根据Cas蛋白组分及氨基酸序列不同，已发现的CRISPR/Cas系统可以分为3种不同类型，Ⅰ型、Ⅱ型和Ⅲ型。其中，Ⅱ型是以Cas9蛋白及导向 RNA 为核心组份，组成较为简单，是目前经过改造用于开发基因组定向编辑技术的主要类型。自CRISPR/Cas9技术体系首先在人类与动物细胞系中建立后，经过改造的 CRISPR/Cas9系统被迅速地应用于拟南芥、烟草、高粱、水稻、小麦、玉米等不同植物基因组的定向编辑研究中。CRISPR/Cas9与ZFNs或TALENs一样都是通过自身的核酸内切酶活性引起靶位点DNA序列双链断裂，然后通过非同源末端连接或同源重组介导的修复2种方式引入突变。至今，在多种作物中已实现诱导产生多种定点突变（包括插入、缺失或修饰等）,并可获得较高的突变诱导率和可稳定遗传的基因组编辑后代植株。与ZFNs或TALENs技术相比，CRISPR/Cas9技术可以实现对基因组中多个靶基因同时进行编辑，从而可以用来修饰同一基因家族中的不同成员或同一代谢途径中的不同调控基因，为其一大优势。由于CRISPR/Cas9技术具有突变诱导率高、成本低、易于操作及可以多重基因编辑等特点，已成为具有广阔应用前景的作物遗传改良与育种研究的分子操作系统。CRISPR技术除了可以对基因组中不同靶基因进行定向编辑以外，还可以广泛地应用于基因表达调控研究、细胞定位运输系统研究及新型RNA沉默系统构建等方面。基因组编辑技术是继转基因技术之后人类对生物进行遗传操作的又一个革命性技术。但是，与转基因技术相比，CRISPR/Cas9基因组编辑技术操作更加简单、快捷。应用 CRISPR/Cas9基因组编辑技术进行育种可以不引入外源基因，在进行基因组编辑之后可以不留下转基因的痕迹，从而导致定义转基因生物的不明确性，因此，政府监管部门是否应该按照转基因的管理办法来监管CRISPR/Cas9技术的应用尚有待决定。
展开▼
