New CRISPR-Cas systems from uncultivated microbes.
New CRISPR-Cas systems from uncultivated microbes.
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DOI：
10.1038/nature21059
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发表时间：
2017-02-09
期刊：
影响因子：
64.8
通讯作者：
Banfield JF
中科院分区：
文献类型：
--
作者：
Burstein D;Harrington LB;Strutt SC;Probst AJ;Anantharaman K;Thomas BC;Doudna JA;Banfield JF
CRISPR-Cas系统通过使用短序列，称为间隔区，引导Cas蛋白切割外来DNA，为微生物提供适应性免疫。2类CRISPR-Cas系统是一种简化的形式，在该系统中，单个结合到RNA上的Cas蛋白识别和切割目标序列。这些最小系统的可编程特性使它们能够重新用途，成为一种广泛革命性的生物和临床研究的多功能技术。然而，目前的CRISPR-Cas技术完全基于分离的细菌的系统，而没有开发来自尚未培养的生物体的绝大多数酶。元基因组学是对从自然微生物群落中提取的DNA进行测序，它提供了获取大量未培养生物的遗传物质的途径。在这里，使用基因组分辨的元基因组学，我们确定了新的CRISPR-CAS系统，包括首次报道的生命古生物领域的Cas9。这种不同的Cas9蛋白是在很少被研究的纳米古生菌中发现的，它是一个活跃的CRISPR-CAS系统的一部分。在细菌中，我们发现了两个以前未知的系统，CRISPR-CasX和CRISPR-Casy，这是迄今发现的最紧密的系统之一。值得注意的是，所有必需的功能成分都通过元基因组学确定，从而能够在大肠杆菌中验证强大的体内RNA引导的DNA干扰活性。结合活体实验对环境微生物群落进行询问，可以获得前所未有的多样性基因组，其内容将扩大基于微生物的生物技术的范围。
CRISPR-Cas systems provide microbes with adaptive immunity by employing short sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA recognizes and cleaves targeted sequences. The programmable nature of these minimal systems has enabled their repurposing as a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving untapped the vast majority of enzymes from organisms that have not been cultured. Metagenomics, the sequencing of DNA extracted from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identified novel CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet identified. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in E. coli. Interrogation of environmental microbial communities combined with in vivo experiments allows access to an unprecedented diversity of genomes whose content will expand the repertoire of microbe-based biotechnologies.