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Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2017-09-21 00:00:00 , DOI: 10.1021/acssynbio.7b00240
S. M. Nayeemul Bari 1 , Forrest C. Walker 1 , Katie Cater 1 , Barbaros Aslan 1 , Asma Hatoum-Aslan 1
Affiliation  

Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.

中文翻译:

使用CRISPR-Cas10编辑强力葡萄球菌噬菌体的策略

葡萄球菌是皮肤上常见的细菌,也是导致抗生素耐药性感染的主要原因。感染和溶解这些生物的病毒(有毒的葡萄球菌噬菌体)可用作常规抗生素的替代品,代表着消除或操纵微生物组中特定物种的有前途的工具。然而,由于其一半以上的基因具有未知的功能,因此有毒的葡萄球菌噬菌体具有固有的风险,可引起未知的下游副作用。此外,它们的迅速和破坏性的生殖周期使它们难以被当前的基因工程技术所困扰。CRISPR-Cas10是精心设计的原核免疫系统,利用小RNA和多亚基蛋白质复合物检测并破坏噬菌体和其他外来核酸。一些葡萄球菌自然拥有CRISPR-Cas10系统,因此,提供了一种已经安装在宿主染色体中的有吸引力的工具,可用于噬菌体基因组工程。然而,尚未探索CRISPR-Cas10对强力葡萄球菌噬菌体的免疫效率以及作为促进其基因组编辑的工具的相应效用。在这里,我们显示了CRISPR-Cas10系统固有的表皮葡萄球菌对多种有毒葡萄球菌噬菌体表现出强大的免疫力。基于此活动,开发了一种通用的两步方法来编辑这些噬菌体,这些噬菌体依赖于宿主中编码的同源重组机制。还介绍了这种方法的变化形式,用于编辑有毒噬菌体基因和访问感染无CRISPR葡萄球菌的噬菌体。这套通用的遗传工具集可以对功能未知的噬菌体基因进行系统研究,并可以设计基于基因的噬菌体基抗菌剂,从而可以消除或操纵特定的葡萄球菌物种。
更新日期:2017-09-21
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