Science2014-06-11 2:30 PM

首次获得Cas9酶三维结构图 Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

论文摘要 

近来,Cas9酶作为一种有力的新基因组编辑工具,引起了人们极大的研究热情。

现在加州大学的生化学家Jennifer Doudna和生物物理学家Eva Nogales领导研究团队,使用X射线晶体衍射技术,获得了两种主要Cas9酶的晶体结构,分辨率达到了2.6和2.2 Å。随后研究团队通过单颗粒电镜向人们展示了,Cas9搭档引导RNA与目标DNA相互作用的机制。这一结果可以帮助人们对Cas9酶进行改良,使其更适合基础研究和基因工程。

“我们在x射线晶体衍射和电镜单颗粒分析的帮助下发现,Cas9蛋白单独存在时处于非活性状态,但与引导RNA结合后,它的三维结构会经历剧烈的改变,允许Cas9与目标DNA结合。” Nogales说。

“获得两种主要Cas9的高分辨率结构之后,我们可以开始理解这类细菌酶的演化,” DouDNA说。“我们看到在催化区域之外,这两种结构差异很大。这种有趣的结构灵活性可以解释Cas9为何能够使用不同种类的引导RNA。我们可以在此基础上设计不影响其功能的小Cas9变体,这对于一些基因组工程应用非常重要。”

细菌一直在和病毒或入侵核酸(质粒)进行斗争,为此它们采用了多种防御机制。以CRISPR序列为核心的适应性核酸免疫系统就是其中之一。在CRISPR和CRISPR相关蛋白(Cas)的帮助下,细菌可以在小rna分子的引导下,靶标和沉默入侵者遗传信息的关键部分。

Cas9是一类受RNA引导的细菌内切酶,被II型CRISPR系统用来识别和剪切特定的双链DNA。人们已经开始利用Cas9在许多真核生物中进行基因组编辑和基因调控。然而,这一技术还没有充分发挥出它的潜力,因为人们还不清楚Cas9靶标DNA的结构基础。

研究人员在链球菌(Streptococcus pyogenes)和放线菌(Actinomyces naeslundii)中解析了Cas9蛋白(SpyCas和AnaCas9)的三维晶体结构。他们发现尽管这些蛋白催化区域外的结构有显著差异,但Cas9家族的成员具有相同的核心结构,这一结构可以裂开两瓣形成钳状。当引导RNA与Cas9结合后,可以使其形成与DNA结合的界面,将Cas9激活。

“我们发现,引导RNA对Cas9转变为活性状态非常重要,”文章的作者之一David Taylor说。“研究显示,我们在使用这一技术时,除了序列互补性,还应道考虑到引导RNA的其它特性。”

Abstract 

Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

Structured Abstract

Introduction
Bacteria and archaea defend themselves against invasive DNA using adaptive immune systems comprising CRISPR (clustered regularly interspaced short palindromic repeats) loci and CRISPR-associated (Cas) genes. In association with Cas proteins, small CRISPR RNAs (crRNAs) guide the detection and cleavage of complementary DNA sequences. Type II CRISPR systems employ the RNA-guided endonuclease Cas9 to recognize and cleave double-stranded DNA (dsDNA) targets using conserved RuvC and HNH nuclease domains. Cas9-mediated cleavage is strictly dependent on the presence of a protospacer adjacent motif (PAM) in the target DNA. Recently, the biochemical properties of Cas9–guide RNA complexes have been harnessed for various genetic engineering applications and RNA-guided transcriptional control. Despite these ongoing successes, the structural basis for guide RNA recognition and DNA targeting by Cas9 is still unknown.

Editor's Summary

Cas9 Solved

Clustered regularly interspaced short palindromic repeats (CRISPR)–associated (Cas) loci allow prokaryotes to identify and destroy invading DNA. Not only important to bacteria, the universal value of Cas endonuclease specificity has also resulted in Cas9 being exploited as a tool for genome editing. Jinek et al. (10.1126/science.1247997, published online 6 February) determined the 2.6 and 2.2 angstrom resolution crystal structures of two Cas9 enzymes to reveal a common structural core with distinct peripheral elaborations. The enzymes are autoinhibited, undergo large conformational changes on binding RNA, and have channels lined with basic residues that are candidates for an RNA-DNA binding groove. Based on these and other insights from the structures, this work provides important revelations both for the CRISPR mechanism and for genome editing.

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