Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis.
Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis.
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DOI：
10.4161/rna.24084
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发表时间：
2013-05
期刊：
影响因子：
4.1
通讯作者：
Terns MP
中科院分区：
文献类型：
--
作者：
Elmore JR;Yokooji Y;Sato T;Olson S;Glover CV 3rd;Graveley BR;Atomi H;Terns RM;Terns MP
CRISPR-Cas系统是RNA引导的免疫系统，可以保护原核生物免受病毒和其他入侵者的侵害。CRISPR基因座编码crRNA，其识别入侵的核酸序列并触发相关Cas蛋白的沉默。存在具有不同组成和机械过程的多种CRISPR-Cas系统。Kodakarensis（Tko）是一种极端嗜热的广古菌，具有I-A型Csa和I-B型Cst CRISPR-Cas系统。我们已经通过RNA深度测序和北方分析分析了来自Tko中的三种CRISPR的crRNA的表达和组成。我们的研究结果表明，与这两个CRISPR-Cas系统相关的crRNA在5′端包含8个核苷酸的保守序列标签。我们用含有内源性crRNA靶向序列的质粒入侵者攻击Tko，并观察到活性CRISPR-Cas介导的沉默。质粒沉默依赖于与crRNA的互补性以及在靶中紧邻crRNA识别位点发现的称为PAM（前间区序列邻近基序）的序列元件。沉默的发生与质粒中靶序列的方向无关，并且似乎发生在DNA水平，推测是通过DNA降解。此外，我们已经通过遗传工程改造染色体CRISPR基因座以表达针对质粒的定制crRNA来定向沉默侵入质粒。我们的研究结果支持CRISPR工程作为一种可行的方法来开发抗感染的原核菌株用于工业。
CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5′ end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry.