Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition.
Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition.
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I-E CRISPR-CAS系统通过与基本配对无关的PAM识别区分了非目标DNA的靶标。
DOI：
10.1371/journal.pgen.1003742
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Brouns SJ
中科院分区：
文献类型：
--
作者：
Westra ER;Semenova E;Datsenko KA;Jackson RN;Wiedenheft B;Severinov K;Brouns SJ
区分自我和非自我是免疫系统的普遍要求。原核生物中的适应性免疫系统以称为CRISPR（成簇的规则间隔短回文重复序列）的重复基因座为中心，入侵者DNA片段被掺入其中。CRISPR转录物被加工成小RNA，其通过互补碱基配对将CRISPR相关（Cas）蛋白引导至入侵核酸。然而，为了避免自身免疫，这些RNA引导物必须专门靶向入侵DNA而不是互补DNA序列（即，自身序列）位于宿主自身的CRISPR基因座中。先前关于来自表皮葡萄球菌的III-A型CRISPR系统的工作已经证明，CRISPR RNA指导序列的一部分参与自我与非自我的辨别。这种自我回避机制依赖于感测RNA引导物和靶DNA侧翼序列之间的碱基配对。为了确定RNA引导物是否参与来自大肠杆菌的I-E型系统中的自我与非自我辨别，我们改变了RNA引导物与DNA靶的侧翼之间的碱基配对潜力。在这里，我们证明了I-E型系统通过一个独立于碱基配对的机制来区分自我和非自我，该机制严格依赖于四个不变的PAM序列的识别。此外，这项工作揭示了引导RNA和紧邻靶序列侧翼的PAM核苷酸之间的第一个碱基对可以被破坏而不影响干扰表型。值得注意的是，这表明该位置的碱基配对不参与外源DNA识别。本文的结果表明，避免自身序列和防止自身免疫的I-E型机制是根本不同的III-A型系统所采用的。我们建议将PAM侧翼序列的排他性靶向称为靶向与非靶向区分机制。CRISPR基因座及其相关基因形成了广泛存在于原核生物中的一组多样的适应性免疫系统。在这些系统中，CRISPR相关基因（cas）编码捕获入侵DNA片段的蛋白质，并将这些序列整合在宿主CRISPR基因座的重复序列之间。这些信息在再次感染时用于降解入侵者基因组。在宿主基因组中储存入侵者序列需要区分入侵者基因组上的入侵者序列和宿主基因组上的入侵者序列的机制。当入侵序列侧翼有重复序列时，表皮葡萄球菌（III-A型系统）的CRISPR-Cas会受到抑制，这会阻止靶向宿主基因组上的CRISPR基因座。在这里，我们证明了大肠杆菌CRISPR-Cas（I-E型系统）不受重复序列的抑制。相反，该系统通过靶中存在真正的原型间隔区邻近基序（PAM）而特异性激活。PAM是与入侵者基因组上的入侵者序列相邻的保守序列，并且这些序列从不与宿主CRISPR基因座内的入侵者序列相邻。PAM识别不受靶标与crRNA的碱基配对潜力的影响。因此，I-E型系统缺乏特异性识别自身DNA的能力。
Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism. CRISPR loci and their associated genes form a diverse set of adaptive immune systems that are widespread among prokaryotes. In these systems, the CRISPR-associated genes (cas) encode for proteins that capture fragments of invading DNA and integrate these sequences between repeat sequences of the host's CRISPR locus. This information is used upon re-infection to degrade invader genomes. Storing invader sequences in host genomes necessitates a mechanism to differentiate between invader sequences on invader genomes and invader sequences on the host genome. CRISPR-Cas of Staphylococcus epidermidis (Type III-A system) is inhibited when invader sequences are flanked by repeat sequences, and this prevents targeting of the CRISPR locus on the host genome. Here we demonstrate that Escherichia coli CRISPR-Cas (Type I-E system) is not inhibited by repeat sequences. Instead, this system is specifically activated by the presence of bona fide Protospacer Adjacent Motifs (PAMs) in the target. PAMs are conserved sequences adjoining invader sequences on the invader genome, and these sequences are never adjacent to invader sequences within host CRISPR loci. PAM recognition is not affected by base pairing potential of the target with the crRNA. As such, the Type I-E system lacks the ability to specifically recognize self DNA.
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影响因子：
16
作者：
Hale, Caryn R.;Majumdar, Sonali;Elmore, Joshua;Pfister, Neil;Compton, Mark;Olson, Sara;Resch, Alissa M.;Glover, Claiborne V. C., III;Graveley, Brenton R.;Terns, Rebecca M.;Terns, Michael P.
通讯作者：
Terns, Michael P.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
3.2
作者：
Deveau, Helene;Barrangou, Rodolphe;Moineau, Sylvain
通讯作者：
Moineau, Sylvain
影响因子：
4.5
作者：
Hale, Caryn;Kleppe, Kyle;Terns, Michael P.
通讯作者：
Terns, Michael P.
通过反式编码的小 RNA 和宿主因子 RNase III 进行 CRISPR RNA 成熟。
影响因子：
64.8
作者：
Deltcheva, Elitza;Chylinski, Krzysztof;Sharma, Cynthia M.;Gonzales, Karine;Chao, Yanjie;Pirzada, Zaid A.;Eckert, Maria R.;Vogel, Joerg;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle