CRISPR-Cas systems restrict horizontal gene transfer in Pseudomonas aeruginosa

CRISPR-Cas systems provide bacteria and archaea with an adaptive immune system that targets foreign DNA. However, the xenogenic nature of immunity provided by CRISPR-Cas raises the possibility that these systems may constrain horizontal gene transfer. Here we test this hypothesis in the opportunistic pathogen Pseudomonas aeruginosa, which has emerged as an important model system for understanding CRISPR-Cas function. Across the diversity of P. aeruginosa, active CRISPR-Cas systems are associated with smaller genomes and higher GC content, suggesting that CRISPR-Cas inhibits the acquisition of foreign DNA. Although phage is the major target of CRISPR-Cas spacers, more than 80% of isolates with an active CRISPR-Cas system have spacers that target integrative conjugative elements (ICE) or the conserved conjugative transfer machinery used by plasmids and ICE. Consistent with these results, genomes containing active CRISPR-Cas systems harbour a lower abundance of both prophage and ICE. Crucially, spacers in genomes with active CRISPR-Cas systems map to ICE and phage that are integrated into the chromosomes of closely related genomes lacking CRISPR-Cas immunity. We propose that CRISPR-Cas acts as an important constraint to horizontal gene transfer, and the evolutionary mechanisms that ensure its maintenance or drive its loss are key to the ability of this pathogen to adapt to new niches and stressors.Subject terms: Microbiology, Microbial genetics, Evolution     

CRISPR-Cas系统在微生物研究中的应用进展

来源于细菌防御系统的CRISPR-Cas系统因其功能强大、多样性受到广泛关注,也在动物、植物、微生物等各个领域得到了应用并迅速发展。概述了CRISPR-Cas系统的作用机理和分类,并对CRISPR-Cas系统在微生物基因编辑和病毒核酸检测方面的研究及应用进展进行了综述,以期为微生物研究工作提供参考。     

CRISPR-Cas13a系统用于肿瘤诊断与治疗的进展

CRISPR-Cas系统是一种目前已知的基因编辑工具,其中以靶向DNA基因组编辑的CRISPR-Cas9系统的研究较为成熟。相较于靶向DNA的基因组编辑技术CRISPR-Cas9系统,近年来靶向RNA的Ⅵ型-CRISPR家族CRISPR-C2c2/Cas13a系统研究日渐增多。CRISPR-Cas13a系统具有特异性识别并结合单链RNA序列从而非特异性切割RNA的特点,可应用于检测肿瘤外周血游离核酸,对早期肿瘤患者进行筛查。同时,Cas13a在进行体内RNA切割的过程中,不涉及编码基因DNA的改变,可直接对基因转录产物mRNA进行编辑,达到基因修饰的目的,并能够同时靶向多基因转录产物从而调控基因的表达。Cas13a系统可应用于分子诊断及RNA编辑中,该系统在肿瘤的诊断与治疗中也被证实具有广阔的发展前景。基于已有的文献资料,文中综述了靶向RNA的CRISPR-Cas13a技术应用于肿瘤诊断与治疗的研究进展,探讨了CRISPR-Cas13a系统对癌症治疗的新思路及存在的局限,并展望了未来可能的研究方向。     

Harnessing the type I CRISPR-Cas systems for genome editing in prokaryotes

Genetic analysis is crucial to the understanding, exploitation, and control of microorganisms. The advent of CRISPR-Cas-based genome-editing techniques, particularly those mediated by the single-effector (Cas9 and Cas12a) class 2 CRISPR-Cas systems, has revolutionized the genetics in model eukaryotic organisms. However, their applications in prokaryotes are rather limited, largely owing to the exceptional diversity of DNA homeostasis in microorganisms and severe cytotoxicity of overexpressing these nuclease proteins in certain genotypes. Remarkably, CRISPR-Cas systems belonging to different classes and types are continuously identified in prokaryotic genomes and serve as a deep reservoir for expansion of the CRISPR-based genetic toolkits. ~90% of the CRISPR-Cas systems identified so far belong to the class 1 system which hinges on multi-protein effector complexes for DNA interference. Harnessing these widespread native CRISPR-Cas systems for ‘built-in’ genome editing represents an emerging and powerful genetic tool in prokaryotes, especially in the genetically recalcitrant non-model species and strains. In this progress review, we introduce the general workflow of this emerging editing platform and summarize its establishment in a growing number of prokaryotes by harnessing the most widespread, diverse type I CRISPR-Cas systems present in their genomes. We also discuss the various factors affecting the success and efficiency of this editing platform and the corresponding solutions.     

The microbial detection array combined with random Phi29-amplification used as a diagnostic tool for virus detection in clinical samples

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples.     

基于CRISPR-Cas13家族的RNA编辑系统及其最新进展

存在于细菌和古菌中的获得性免疫系统CRISPR-Cas目前已被广泛应用到生物技术领域,尤其是靶向DNA的CRISPR-Cas9技术。然而CRISPR-Cas系统靶向RNA的技术还处于初步应用阶段。Ⅵ型CRISPR-Cas系统(CRISPR-Cas13)的发现,揭示了RNA引导的RNA靶向性。CRISPR-Cas13是目前CRISPR-Cas家族中唯一只靶向ssRNA的系统,为RNA靶向和RNA编辑奠定了基础。根据Cas13系统发育已证明将Ⅵ型CRISPR-Cas系统分为4种亚型(A-D)。主要对目前最新的靶向RNA技术的CRISPR-Cas13家族的分类以及防御机制进行了综述,介绍了 CRISPR-Cas13 技术的应用以及基于CRISPR-Cas13家族的RNA编辑系统的最新研究进展。最后,对目前CRISPR-Cas13 RNA编辑技术体系存在的问题进行了分析和对未来的发展进行展望。     

Highly sensitive detection of Staphylococcus aureus directly from patient blood

BackgroundRapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing – polymerase chain reaction (PCR) platform as a model diagnostic system.ConclusionsWe have demonstrated a highly sensitive two-hour assay for detection of sepsis causing bacteria like S. aureus directly in 1 ml of whole blood, without the need for blood culture.     

Microbial diagnostic microarray for food‐ and water‐borne pathogens

A microbial diagnostic microarray for the detection of the most relevant bacterial food- and water-borne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labelling of oligonucleotides and the pyhylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus/species level) and sensitive (0.1% relative and 10⁴ cfu absolute detection sensitivity) detection of the target pathogens. Validation was performed using a set of reference strains and a set of spiked environmental samples. Reliability of the obtained data was additionally verified by independent analysis of the samples via fluorescence in situ hybridization (FISH) and conventional microbiological reference methods. The applicability of this diagnostic system for food analysis was demonstrated through extensive validation using artificially and naturally contaminated spiked food samples. The microarray-based pathogen detection was compared with the corresponding microbiological reference methods (performed according to the ISO norm). Microarray results revealed high consistency with the reference microbiological data.     

CRISPR-Cas系统及anti-CRISPR蛋白在微生物中的研究进展

CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISP) and CRISPR associated proteins)系统是细菌用来防御病毒、质粒等外源核酸入侵的一种获得性免疫防御系统。随着研究的深入,CRISPR-Cas系统已发展为一种重要的基因编辑工具,并成功应用于动物、植物和微生物的基因改造中。但该基因编辑方法有时存在基因脱靶效应,从而限制了其推广应用。最近,通过将1种新发现的抗CRISPR蛋白(Anti-CRISPR protein,ACP)与CRISPR-Cas系统相结合,已成功开发出可控制基因脱靶效率的CRISPR-Cas基因编辑工具。本文首先对CRISPR-Cas系统及ACP进行了简要介绍,然后就CRISPR-Cas基因编辑工具及ACP在微生物基因改造的应用现状进行了综述,并对ACP介导的CRISPR-Cas基因编辑方法(ACP-CRISPR-Cas)在微生物基因编辑中的应用前景进行了讨论。     

细菌CRISPR-Cas系统的研究进展

获得性免疫长期以来被视为真核生物所独有,而CRISPR-Cas系统的发现则打破了这一定论。它是广泛存在于细菌和古菌中的一种获得性免疫系统,通过捕获整合初次感染的外源核酸片段,在Cas蛋白与cr RNA(CRISPR RNAs)的共同作用下抵御相同核酸的再次入侵,以保护宿主免受侵扰。近些年,CRISPR-Cas系统得到广泛的关注和研究。本文主要从细菌微生物角度,对系统分类、作用机制及原核领域应用等取得重要突破的研究进行扼要阐述,为CRISPR-Cas系统的深入探究和应用拓展提供有价值的参考信息。     

基于CRISPR/Cas体系的生物传感器在病原体核酸检测方面的应用

成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列相关蛋白 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein,CRISPR/Cas) 因高效的靶向结合和可编程性,已被开发为一种精准、高效、低价和高灵敏度的核酸检测工具。目前基于CRISPR-Cas体系的生物传感器在病原体核酸检测方面显示出了优良的性能,受到了人们的广泛关注,这种新型的病原体核酸检测有望替代传统的病原体检测方法。文中就基于CRISPR/Cas体系的生物传感器在病原体核酸检测中的最新研究进展进行综述。     

Cas9HF1 enhanced specificity in Ustilago maydis

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.     

基于纳米信号标签的表面增强拉曼散射在病原菌检测中的应用

表面增强拉曼散射(surface-enhanced Ranan scattering,SERS)技术由于其灵敏度高、检测速度快、高特异性和无损等优点,在病原菌检测领域受到了广泛的关注.主要总结了近年来基于纳米信号标签的SERS方法在检测病原菌领域中的研究进展,并介绍了多功能SERS检测平台的构建及在病原菌检测中的应用....     

Epidemiological and evolutionary consequences of different types of CRISPR-Cas systems

Bacteria have adaptive immunity against viruses (phages) in the form of CRISPR-Cas immune systems. Currently, 6 types of CRISPR-Cas systems are known and the molecular study of three of these has revealed important molecular differences. It is unknown if and how these molecular differences change the outcome of phage infection and the evolutionary pressure the CRISPR-Cas systems faces. To determine the importance of these molecular differences, we model a phage outbreak entering a population defending exclusively with a type I/II or a type III CRISPR-Cas system. We show that for type III CRISPR-Cas systems, rapid phage extinction is driven by the probability to acquire at least one resistance spacer. However, for type I/II CRISPR-Cas systems, rapid phage extinction is characterized by an a threshold-like behaviour: any acquisition probability below this threshold leads to phage survival whereas any acquisition probability above it, results in phage extinction. We also show that in the absence of autoimmunity, high acquisition rates evolve. However, when CRISPR-Cas systems are prone to autoimmunity, intermediate levels of acquisition are optimal during a phage outbreak. As we predict an optimal probability of spacer acquisition 2 factors of magnitude above the one that has been measured, we discuss the origin of such a discrepancy. Finally, we show that in a biologically relevant parameter range, a type III CRISPR-Cas system can outcompete a type I/II CRISPR-Cas system with a slightly higher probability of acquisition.