中国农业科学 ›› 2015, Vol. 48 ›› Issue (9): 1669-1677.doi: 10.3864/j.issn.0578-1752.2015.09.01

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

CRISPR/Cas9系统在植物基因组定点编辑中的研究进展

解莉楠,宋凤艳,张旸   

  1. 东北林业大学生命科学学院,哈尔滨 150040
  • 收稿日期:2014-11-12 出版日期:2015-05-01 发布日期:2015-05-01
  • 通讯作者: 张旸,Tel:0451-82191783;E-mail:summerzhang@126.com
  • 作者简介:解莉楠,Tel:0451-82191783;E-mail:linanxie@126.com
  • 基金资助:
    黑龙江省自然科学基金(C201343)、中央高校基本科研业务费专项资金(2572014CA21)、哈尔滨市科技创新人才研究专项资金(2013RFQXJ036)、黑龙江省博士后资助(LBH-Z14009)、“十二五”863计划现代农业技术领域项目(2011AA10020801,2013AA102706)

Progress in Research of CRISPR/Cas9 System in Genome Targeted Editing in Plants

XIE Li-nan, SONG Feng-yan, ZHANG Yang   

  1. College of Life Science, Northeast Forestry University, Harbin 150040
  • Received:2014-11-12 Online:2015-05-01 Published:2015-05-01

摘要: 当外源DNA通过转基因技术导入植物细胞后,会以同源重组或非同源重组两种不同的方式整合到基因组中,进而获得相应的目标性状。外源DNA与受体细胞序列相同或相近的位点发生重新组合,从而整合到受体细胞的染色体上称之为同源重组;当发生了DNA双链断裂的细胞为了避免DNA或染色体断裂而造成DNA降解或对生命力的影响,而强行将2个DNA断端彼此连接在一起时则为非同源重组。发生非同源重组的细胞其基因组常出现核苷酸片段的插入和/或缺失以及其他突变等多种情况,使得研究者无法得到精确控制的突变结果;而发生同源重组的细胞基因组序列通常不变,通过加入同源重组的供体DNA,可以实现对基因组的精确修饰和改造。由于在植物中产生自发同源重组的概率很低,对植物基因组进行精确修饰和改造非常困难,位点特异性核酸酶的出现和应用,大大提升了同源重组的效率,使基因组编辑变得更加高效和精确,从而使得对包括植物在内的任何物种进行基因组编辑都将成为可能。锌指核酸酶(ZFN)和TALE核酸酶(TALENs)是能够使DNA的靶位点产生DNA双链断裂进而实现基因组定点编辑的常用系统,但在具体应用中发现这两种系统存在着许多缺陷和不足,如脱靶效应、与基因组进行特异结合与染色体位置及邻近序列有关等,另外技术难度大、构建组装时间长也限制了其应用。CRISPR/Cas系统广泛存在于细菌及古生菌中, 是机体长期进化形成的RNA指导的降解入侵病毒或噬菌体DNA的适应性免疫系统。Ⅱ型CRISPR/Cas系统经过密码子优化等改造后已成为继锌指核酸酶ZFNs和TALENs后的新型高效定点编辑的新技术,具有突变效率高、制作简单、易操作及成本低的特点。目前,该技术成功应用于人类细胞、斑马鱼、小鼠以及细菌的基因组精确编辑,编辑的类型包括基因的定点插入、小片段的缺失、多个位点同时突变、基因定点的indel突变等。目前,CRISPR/Cas系统在植物中的应用还比较有限,但该技术为植物基因工程的发展呈现了美好的前景。文中首先简要介绍了CRISPR/Cas系统的组成和基本原理,进而详细综述了该技术在植物内源基因和外源基因定点编辑中的应用,主要列举了自CRISPR/Cas系统改造成功以来利用该系统对单子叶和双子叶植物进行基因组定点编辑的案例,最后对基因组编辑技术在农业和植物基因工程上的应用进行了展望,希望能够为开展该领域研究的科研工作者提供参考。

关键词: CRISPR/Cas系统, 基因组编辑, 植物基因工程

Abstract: When exogenous DNA was imported into plant cell by transgenic technology, DNA fragment will integrate into the genome by homologous recombination or nonhomologous recombination. In addition, the plants seedling will achieve corresponding target traits. Homologous recombination occurred when the exogenous DNA and the same or similar sequences in receptor cells recombined and integrated to the receptor cell’s chromosomes, so the sequence will be possible to precisely modified and transformed. However, in some cases, to avoid the fracture caused by DNA or chromosomal DNA degradation or the impact on the vitality, the two double-stranded DNA break ends will be joined without considering the sequence similarity by error-prone nonhomologous end joining. As a result, precise mutations control is more difficult to achieve because insertion and/or deletion and other variety of mutations are high-frequently occurred in non-homologous recombination than in homologous recombination. Unfortunately, the frequency of homologous recombination is very low in plants which results the undesirable genome editing. Site-specific nucleases make genome editing more efficient and more precise by the great improvement of the efficiency in the homologous recombination. Such nucleases, zinc finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been demonstrated to efficiently produce a DNA double-strand break at target site and to induce genome modification in a variety of organisms including plants. However, some defects found in the specific application, such as off-target effects, specific combined site with genome affected by chromosomal location and adjacent sequence, furthermore, technical complexity and time-consuming for assemble limit its application. The clustered, regularly interspaced, short palindromic repeats(CRISPR) system is a prokaryotic adaptive immune system which widely exists in bacteria and archaea. As the result of the long-term evolution, the system can defense against the degradation of RNA to guide the invasion of the virus or phage DNA. Recent advances in the study demonstrated CRISPR/Cas typeⅡ system was a promising system of genome editing strategy with high efficiency, affordability and easy to engineer compared with ZFNs and TALENs. Many precise genome editing cases by CRISPR/Cas system were found successfully in human cells, zebra fish, mice and bacterial, including gene insertion, deletion, mutation in multiple sites or in specific locus. Although the application in plants is still in a relatively limited range, there is an outstanding prospect of CRISPR/Cas system in plant genetic engineering. A brief summary of the composition and principles was presented firstly. Then, the authors emphasis on citing numerous cases involved its application of exogenous and endogenous genes editing in cotyledon and dicotyledonous plants which demonstrate that the CRISPR/Cas9 system has become a powerful tool in plant genome engineering. Finally, the future of the genome technology application in agriculture and plant genetic engineering was discussed which will provide a reference for researchers in genetic modification.

Key words: CRISPR/Cas system, genome editing, plant genetic engineering