DNA targeting by the type I-G and type I-A CRISPR-Cas systems of Pyrococcus furiosus.
DNA targeting by the type I-G and type I-A CRISPR-Cas systems of Pyrococcus furiosus.
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DOI：
10.1093/nar/gkv1140
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发表时间：
2015-12-02
影响因子：
14.9
通讯作者：
Terns MP
中科院分区：
文献类型：
--
作者：
Elmore J;Deighan T;Westpheling J;Terns RM;Terns MP
CRISPR-Cas系统沉默原核生物中的质粒和病毒。CRISPR- Cas效应复合物含有CRISPR rna (crrna)，包括从入侵者和直接CRISPR相关（Cas）蛋白中捕获的序列，以破坏相应的入侵者核酸。发热焦球菌（Pfu）拥有三种CRISPR-Cas免疫系统：一个Cst （I-G型）系统在一个位点上具有相关的Cmr （III-B型）模块，另一个位点上具有部分Csa （I-A型）模块（缺乏已知的入侵者序列获取和crRNA加工基因）。Pfu Cmr复合物可切割互补的靶rna， Csa系统已被证明可靶向DNA，而Cst复合物沉默入侵者的机制尚不清楚。在这项研究中，我们研究了Cst和Csa系统在携带单个CRISPR-Cas系统的Pfu菌株中的功能。质粒转化实验表明，Cst和Csa系统都通过DNA沉默发挥作用，并利用相似的侧翼序列信息（PAMs）识别入侵DNA。每个系统的沉默都需要其相关的Cas3核酸酶。来自Pfu中7个共享CRISPR位点的crRNA被所有3个效应复合体加工使用，Northern分析显示单个效应复合体决定了产生的成熟crRNA物种的特征。
CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated.