《植物生理学报》 2015, 51(12): 2063-2069

基因组编辑新技术: CRISPR/Cas系统在生物基因组学中的研究进展

吴彩云, 罗秉轮, 孙玉强^*

杭州师范大学生命与环境科学学院, 杭州310036

通信作者：孙玉强；E-mail: sunyuq1109@hotmail.com；Tel: 0571-28862816

摘　要：

近两年, CRISPR/Cas技术的发现和应用, 迅速丰富和发展了基因组编辑技术, 并以此演化更多基因组研究的手段。基因组编辑是指将外源DNA元件导入到细胞染色体特定位点, 定点改造基因组, 获得预期的生物体基因组序列发生遗传改变的技术, 包括通过基因敲入(knock-in)、基因敲除(knock-out)定点改造基因组从而改变基因组中的特定基因、基因表达方式或调控其它基因表达模式等, 达到研究基因功能的目的。目前, 基因组编辑技术主要有锌指核酸酶(ZFNs)、转录激活因子样效应物核酸酶(TALENs)和CRISPR/Cas系统。CRISPR/Cas系统是细菌及古细菌在进化过程中形成, 主要针对侵入的噬菌体等生物外源基因在体内整合或切除从而产生获得性免疫系统。根据出现特定CRISPR蛋白编码基因相邻位点重复数,CRISPR/Cas系统分为三种类型, 系统I和III目前不适合应用。而系统II最为简单, 应用最广泛, 由crRNA、tracrRNA以及Cas9蛋白组成。本文主要讨论最新的基因组编辑技术CRISPR/Cas系统的结构特点、作用原理及在基因组定点修饰方面的应用, 并对该技术运用中所遇到或可能遇到的脱靶等问题及潜在的应用前景进行分析。

关键词：基因组编辑; 同源重组; CRISPR/Cas; 脱靶效应

收稿：2015-07-13   修定：2015-10-09

资助：浙江省自然科学基金(LR14C130001)和杭州市科委种子种苗专项(20140932H10)。

A New Genome Edited Technology: CRISPR/Cas System and Its Research Progress in Biological Genomics

Wu Cai-Yun, Luo Bing-Lun, Sun Yu-Qiang^*

College of Life and Environmental Science, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China

Corresponding author: Sun Yu-Qiang; E-mail: sunyuq1109@hotmail.com; Tel: 0571-28862816

Abstract:

In past two years, CRISPR/Cas system works as a new genome editing technology for its unique advantages and specialty, quickly enriches and promotes the genome editing technologies, and was applied rapidly in various species. More techniques were created on the base of CRISPR/Cas system for genome research. Genome editing refers that the foreign DNA components imported to the specific loci at chromosomes in cells to modify target genome for obtaining the desired genetic changes. There are many ways for studying gene expression pattern, such as gene knockout and knock in, which are the conventional ways to study function of the target genes. Recently, genome editing technologies mainly included ZFNs, TALENs and CRISPR/Cas system. CRISPR/Cas system originated from the acquired immunity of bactericas and archaebacteria, which used to defense against viruses and plasmids infecting by targeting the specific nucleic acid sequence. Based on the presence of genes that coded specific Cas protein located in close proximity to the repetitive arrays, CRISPR/ Cas was classified into three groups. Type I and III required multiple proteins to find and cut target DNA, they are not readily applicated. In contrast, type II CRISPR system is simple and popularly used for just easily obtaining crRNA, tracrRNA and Cas9 protein. We discussed here on the structure, function, mechanism and applications of CRISPR/Cas, and further discussed the challenges as off-target and prospects.

Key words: genome editing; homologous recombination (HR); CRISPR/Cas; off-target