CRISPR/Cas9介导的基因编辑技术研究进展

Advances in CRISPR/Cas9-mediated gene editing

CRISPR/Cas9(Clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9)是最近发现的一种新型的基因组定点编辑技术。CRISPR即成簇的、有规律的、间隔短回文重复序列,是人们在研究大肠杆菌编码的碱性磷酸酶基因时发现的,它原本是存在于细菌和古细菌基因组中含有多个短重复序列的基因位点,能够为自身提供一种特异性免疫保护机制,抵御外来病毒、质粒等遗传元件的入侵。CRISPR系统主要依赖cr RNA(CRISPR RNA)和tracr RNA(Trans-activating chimeric RNA)结合并导向Cas(CRISPR-associated system)蛋白来对外源DNA进行序列特异性降解。目前已经发现了3种类型的CRISPR/Cas系统:Ⅰ型、Ⅱ型和Ⅲ型。其中Ⅱ型系统组分较为简单,主要依赖的是Cas9核心蛋白,在RNA的介导下,Cas9蛋白能够识别靶序列进行切割造成DNA的双链断裂(Double-strand breaks,DSB)。在此基础上,人们可以对基因组的特定位点进行基因打靶、基因定点插入、基因修复等各种遗传操作。这种新的基因组定点编辑技术比类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)和锌指核酸酶(Zinc-finger nuclease,ZFN)技术设计更加简单、更容易操作,势必会有更广泛的应用。目前,利用该技术已在多个物种的细胞和个体水平上实现了遗传操作。文中将从CRISPR/Cas9的来源、结构、作用机理方面介绍其研究进展,为开展这一领域的研究工作提供参考。

Clustered regulatory interspaced short palindromic repeats（CRISPR） found in bacteria and archaea genome that contains multiple short repeats loci, provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage. The first inkling of this hot new genetic engineering tool turned up in 1987, when a research team observed an oddly repetitive sequence at one end of a bacterial gene. Now three types of CRISPR/Cas system have been identified： types Ⅰ, Ⅱ and Ⅲ. In the type Ⅱ CRISPR/Cas9 system, short segments of foreign DNA termed ‘spacers＇ are integrated within the CRISPR genomic loci, transcribed and processed into short CRISPR RNA（cr RNA）. These cr RNAs anneal to trans-activating cr RNA（tracr RNA） and direct sequence-specific cleavage in that a double-strand break（DSB） is generated by Cas proteins. Based on these findings, various genetic methods, including gene targeting（Gene disruption）, gene insertion, gene correction etc., are being designed to manipulate the genomes of different species at specific loci. Compared with zinc finger nucleases（ZFN） and transcription activator-like effector nucleases（TALEN）, CRISPR/Cas9 is simpler with higher specificity and less toxicity. This review summarizes recent progress, discusses the prospects of CRISPR/Cas9 system, with an emphasis on its structure, principle, applications and potential challenges, and provides a useful reference for researchers who are interested in this new technique.