【3篇Nature、1篇Cell】重大突破!CRISPR再现“魔剪”实力,这次只“剪”单个碱基
2016/04/23
4月20日,在线发布于Nature杂志上的一项研究中,科学家们报告称,一种经改造的CRISPR基因编辑系统能够赋予研究者们有效改变给定基因中单个DNA碱基的能力。


4月20日,Nature杂志在线发表了3篇CRISPR相关的研究进展。在题为“Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage”的研究中,科学家们报告称,一种经改造的CRISPR基因编辑系统能够赋予研究者们有效改变给定基因中单个DNA碱基的能力。这一研究进展进一步开拓了基因编辑的应用范围,且有望为开发多种疾病的治疗方法带来新的可能。

近几年,CRISPR–Cas9基因编辑系统被全球科学家们广泛应用,然而尽管很容易用它改变基因的功能,但是修复点突变一直是个未解的难题。该研究的通讯作者、哈佛大学化学生物学家David Liu说:“这些点突变非常重要,事实证明,大多数与遗传变异相关的疾病都是点突变。现有的遗传方法对修复点突变都不是非常有效。”


如何实现只“剪”一个碱基

Cas9酶的作用机制是在导向RNA分子的引导下靶向基因组中的特定位点。一旦Cas9酶发现与导向RNA匹配的DNA序列,它会切断DNA双链。细胞对此的反应是修复“缺口”,插入或删除碱基;但这一修复过程往往非常“草率”,最终的结果通常是产生一个“残疾”的基因。研究人员鼓励的一种修复模式是,通过提供一个能让细胞用作指导的DNA模板,从而使修复酶能够在缺口处插入匹配的DNA序列。但这种方法成功的时间不到5%,甚至更少。

在这一研究中,科学家们找到一种不用切割就能编辑基因组的方法。他们改造了Cas9酶,使它不再能够切割DNA;然后将其与另一种能够将一种碱基(胞嘧啶,C)转变为另一种碱基(尿嘧啶,U)的酶绑定在一起。U通常存在于RNA中,在DNA中细胞会把它当作T来阅读。在这样一个系统中,导向RNA将经改造的Cas9送到基因组中的目标位置,系统中的另一种酶(碱基修饰酶APOBEC1)发挥改变DNA序列的作用,而不是去切断它们。

结果发现,经改造的这一系统在试管中对单独DNA的作用约达44%的时间,这是一个很大的提高;但在细胞中,最好的效率也只有7.7%。一个问题是,这一系统只是改变了其中一条DNA链,会使其与另一条链不匹配。此时,细胞会开始修复这种“错误匹配”,抵消酶系统的作用。

随后,Liu的团队开始添加另一种蛋白到经改造的Cas9上,用于阻止细胞从DNA上去除U。最终产生的这一碱基编辑器(base editor)纠正了小鼠细胞中阿尔茨海默病相关的突变,有效率高达75%。同时,这一碱基编辑器还纠正了人类细胞中癌症相关基因的突变,效率高达7.6%。此前,标准的CRISPR-Cas9方法从未做到这样。

Liu说,还有成百上千的其它疾病相关的突变可能通过“C to U”开关纠正。不过,团队目前正努力将这种方法扩展到其它点突变,并尝试在动物模型中使用这一技术。他希望,这一系统能够使创建携带人疾病相关基因突变的小鼠模型更容易,最终,在经过多年的测试和改进后,能够用于疾病治疗。


张锋等科学家Cell发表CRISPR-Cpf1新成果

如今,在CRISPR系统中,Cas9酶已经不是唯一。去年9月25日,发表在Cell杂志上的一项研究中,张锋团队发现一种叫做Cpf1的蛋白可能将克服CRISPR-Cas9系统应用中的一些限制。科学家们评估了来自16种不同细菌的Cpf1酶,最终发现有2种Cpf1能够剪切人类DNA。Nature杂志表示,Liu等人的结果也许也可以用于CRISPR系统家族中的其它酶。

4月21日,发表在《细胞》杂志上的题为“Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA”的研究中,来自Broad研究所和东京大学的研究人员揭示出了Cpf1/向导RNA/靶DNA复合物的晶体结构。张锋及东京大学的Osamu Nureki教授是这一研究的共同通讯作者。

该研究中,科学家们确定了氨基酸球菌属(Acidaminococcus sp)Cpf1(AsCpf1)与向导RNA和靶DNA复合物的晶体结构,分辨率达到2.8 埃(Å)。AsCpf1采用了一种二裂片(bilobed)结构,RNA-DNA异源双链核酸分子束缚在中间通道内。他们通过比较AsCpf1和Cas9的结构,揭示出一些惊人的相似性和主要的差异,由此解释了它们独特的功能。AsCpf1包含RuvC结构域和一个推定的新核酸酶结构域,它们分别负责切割非靶向及靶向DNA链,由此生成交错的DNA双链断裂。AsCpf1借助了一些碱基和形状读取机制来识别5′-TTTN-3′PAM。

中国科学家破译CRISPR-Cpf1运行机制


黄志伟教授和课题组成员在实验室(图片来源:哈尔滨工业大学官网)

与Liu等科学家的研究结果同期发表的另外两篇Nature也与这一有望代替Cas9的Cpf1酶有关。在题为“The crystal structure of Cpf1 in complex with CRISPR RNA”的研究中,哈尔滨工业大学生命学院黄志伟教授及其团队首次揭示了CRISPR-Cpf1识别crRNA的复合物结构。

研究通过结构生物学和生化研究手段揭示了CRISPR-Cpf1识别CRISPR RNA以及Cpf1剪切pre-crRNA成熟的分子机制,这对认识细菌如何通过CRISPR系统抵抗病毒入侵的分子机理具有十分重要的科学意义。研究还发现,Cpf1并不是此前人们推测的二聚体状态,它本身是一个呈三角形的单体,位于该结构中间是一个带有正电荷的凹槽。这些研究为成功改造Cpf1系统,使之成为特异的、高效的全新基因编辑系统提供了结构基础。

据介绍,Cpf1和Cas9很大的不同在于:Cpf1仅需要一个拷贝的crRNA,而Cas9需要序列更长的tracrRNA和crRNA去识别、剪切底物DNA,较短的crRNA在转染细胞过程中将更高效。

该研究发现Cpf1在没有crRNA结合的状态下处于松散的构象,crRNA的结合引起Cpf1发生显著的构象变化。与Cas9结合sgRNA极为不同的是,仅仅crRNA的重复序列部分(repeat sequence)就能引起Cpf1构象的巨大变化,这反映了这类短小的crRNA与Cas9结合的长sgRNA的识别机制的巨大差别。该结构显示来自于H843、 K852以及K869催化残基侧链上的氮原子位于一个平面上,同时和RNA A(+20)的磷酸基团形成氢键,该结构证据表明Cpf1剪切pre-crRNA成为crRNA是一个碱催化的反应。

CRISPR先驱Emmanuelle Charpentier新成果

在题为“The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA”的研究中,CRISPR“两大女神”之一的Emmanuelle Charpentier领导的科学家小组证明了Cpf1在定向基因编辑中能执行RNA加工和DNA切割两个活动,这可能打开了对特定序列基因组编辑和沉默的新方向。

备注:本文部分内容编译自Nature,部分内容参考自生物通、黑龙江日报、哈尔滨工业大学官网、中国科学报。

推荐阅读

Gene-editing hack yields pinpoint precision

黄志伟团队在《自然》发表论文揭示CRISPR-Cpf1识别crRNA以及剪切pre-crRNA的机制

张锋Cell重大突破:基因编辑家族“新神器”诞生,CRISPR/Cpf1让一切皆有可能!

所有文章仅代表作者观点,不代表本站立场。如若转载请联系原作者。
查看更多
  • The crystal structure of Cpf1 in complex with CRISPR RNA

    The CRISPR–Cas systems, as exemplified by CRISPR–Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection1, 2, 3, 4, 5, 6, 7. The CRISPR–Cpf1 system, a new class 2 CRISPR–Cas system, mediates robust DNA interference in human cells1, 8, 9, 10. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)2+ ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.

    展开 收起
  • Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

    Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction1, 2. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks1, 2. Here we report the development of ‘base editing’, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting ‘base editors’ convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15–75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

    展开 收起
  • The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA

    CRISPR–Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA1. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA)2, 3, 4, 5 and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA6, 7, 8, 9, 10, 11, 12. In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA–crRNA duplex then guides Cas9 to cleave target DNA15. Here, we demonstrate a novel mechanism in CRISPR–Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5′-YTN-3′ protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5′ overhang16. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR–Cas systems so far described.

    展开 收起
  • Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA

    Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA-DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for cleaving the non-target and target strands, respectively, and for jointly generating staggered DNA double-strand breaks. AsCpf1 recognizes the 5′-TTTN-3′ protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1 and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox. To access this article, please choose from the options below

    展开 收起
发表评论 我在frontend\modules\comment\widgets\views\文件夹下面 test