基于CRISPR/Cas系统的核酸生物传感器

近年来,CRISPR/Cas系统已经成为转录调控和基因组编辑的重要工具。除了在基因编辑领域的贡献,CRISPR/Cas系统独特的靶核酸顺式切割和非特异性单链核酸反式切割能力,在开发核酸检测的新型生物传感器方面展现出巨大潜力。构建基于CRISPR/Cas系统高灵敏度生物传感器的关键通常依赖其与不同信号扩增策略,诸如核酸扩增技术或特定信号转导方法的结合。基于此,本文旨在通过介绍不同类型的CRISPR/Cas系统,全面概述基于该系统的核酸检测生物传感器的研究进展,并重点对结合核酸扩增技术（PCR、LAMP、RCA、RPA和EXPAR）、灵敏的信号转导方法（电化学和表面增强拉曼光谱）和特殊结构设计生物传感的三大类型信号放大策略的CRISPR/Cas生物传感器进行总结和评论。最后,本文对目前的挑战以及未来的前景进行展望。     

基于CRISPR/Cas技术的核酸检测研究进展

由成簇、规则间隔的短回文重复序列（Clustered regularly interspaced short palindromic repeats,CRISPR）和CRISPR相关蛋白（CRISPR-associated protein,Cas）组成的CRISPR/Cas系统是广泛存在于多数细菌和古细菌中的一种适应性免疫系统。CRISPR/Cas系统可识别并结合外源入侵的核酸分子,之后Cas蛋白的切割活性被激活,能够对入侵的核酸分子进行切割使其降解。利用CRISPR/Cas系统特异的序列识别及切割活性,将其应用于核酸检测中,为提高检测灵敏度及特异性等性能指标提供了一种新思路。本文介绍了CRISPR/Cas系统的发展、作用机制等,对多样化的Cas蛋白在核酸检测中的代表性应用研究进行总结,进一步讨论了CRISPR/Cas技术应用于核酸检测中存在的优缺点,并对未来研究进行了展望,为基于CRISPR/Cas技术的核酸检测方法在病原微生物的检测中提供参考和依据。     

Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.     

Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.     

基于CRISPR/Cas的分子诊断传感器在非核酸分子诊断中的应用

CRISPR/Cas不仅是一种重要的基因编辑工具,而且还是一种有效的分子诊断工具。目前基于CRISPR/Cas建立了一系列的分子诊断传感器系统,广泛应用于核酸、非核酸等检测过程中。与应用较广泛的核酸分子诊断传感器系统相比,基于CRISPR/Cas的非核酸检测系统目前尚未见系统性综述,因此本文围绕基于CRISPR/Cas12和CRISPR/Cas13建立的两大类非核酸分子传感器诊断系统的基本特征、工作流程及其检测原理等进行了全面综述,期望能为CRISPR/Cas分子诊断系统在体外诊断中的应用提供依据。     

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。     

Size-dependent chromatographic separation of nucleic acids

Chromatographic procedures currently used for the size-dependent fractionation of nucleic acids are reviewed. First, an attempt is made to clarify the concept of “size” of nucleic acids and then various aspects of the chromatography of nucleic acids are considered. It is emphasized that consideration of the dynamic three-dimensional structure of large polynucleotides in a rapidly flowing eluent is essential for both the better understanding of mechanism and the development of sophisticated procedures. Of the practical chromatographic techniques that are not based on true size fractionation, ion-exchange chromatography on non-porous column packings appears to be the most efficient. Other methods, such as hydrophobic interaction, are unlikely to become popular. As for truly size-dependent modes, there are gel permeation and slalom chromatography. Although media with extremely large pores become available, the efficiency of gel permeation is still low as a practical separation procedure for large nucleic acid molecules. Its best use seems to be in the field of physicochemical research into nucleic acids in solution. The newly discovered slalom chromatography is based on a principle completely different from all other chromatographic modes. It enables the efficient separation of large double-stranded DNA fragments of 5–50 kilo base pairs by discriminating their length. It has proved not only to be useful as a tool for nucleic acid research but also to have great significance in other fields, e.g. the hydrodynamics of polymer solutions, the search for new chromatographic modes, etc.     

Structural principles of CRISPR-Cas enzymes used in nucleic acid detection

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-based technology has revolutionized the field of biomedicine with broad applications in genome editing, therapeutics and diagnostics. While a majority of applications involve the RNA-guided site-specific DNA or RNA cleavage by CRISPR enzymes, recent successes in nucleic acid detection rely on their collateral and non-specific cleavage activated by viral DNA or RNA. Ranging in enzyme composition, the mechanism for distinguishing self- from foreign-nucleic acids, the usage of second messengers, and enzymology, the CRISPR enzymes provide a diverse set of diagnosis tools in further innovations. Structural biology plays an important role in elucidating the mechanisms of these CRISPR enzymes. Here we summarize and compare structures of three types of CRISPR enzymes used in nucleic acid detection captured in their respective functional forms and illustrate the current understanding of their activation mechanism.     

Evolution of animal Piwi-interacting RNAs and prokaryotic CRISPRs

Piwi-interacting RNAs (piRNAs) and CRISPR RNAs (crRNAs) are two recently discovered classes of small noncoding RNA that are found in animals and prokaryotes, respectively. Both of these novel RNA species function as components of adaptive immune systems that protect their hosts from foreign nucleic acids-piRNAs repress transposable elements in animal germlines, whereas crRNAs protect their bacterial hosts from phage and plasmids. The piRNA and CRISPR systems are nonhomologous but rather have independently evolved into logically similar defense mechanisms based on the specificity of targeting via nucleic acid base complementarity. Here we review what is known about the piRNA and CRISPR systems with a focus on comparing their evolutionary properties. In particular, we highlight the importance of several factors on the pattern of piRNA and CRISPR evolution, including the population genetic environment, the role of alternate defense systems and the mechanisms of acquisition of new piRNAs and CRISPRs.     

Ionic liquids: prospects for nucleic acid handling and delivery

Operations with nucleic acids are among the main means of studying the mechanisms of gene function and developing novel methods of molecular medicine and gene therapy. These endeavours usually imply the necessity of nucleic acid storage and delivery into eukaryotic cells. In spite of diversity of the existing dedicated techniques, all of them have their limitations. Thus, a recent notion of using ionic liquids in manipulations of nucleic acids has been attracting significant attention lately. Due to their unique physicochemical properties, in particular, their micro-structuring impact and tunability, ionic liquids are currently applied as solvents and stabilizing media in chemical synthesis, electrochemistry, biotechnology, and other areas. Here, we review the current knowledge on interactions between nucleic acids and ionic liquids and discuss potential advantages of applying the latter in delivery of the former into eukaryotic cells.     

Visualization of viral assembly in the infected cell.

The study of the virus life cycle in infected cells is a methodological challenge due to the small size and diversity of the viral components. Recent developments on preservation of fine structure and molecular localization have provided a group of powerful methods with wide applications in cell biology and virology. Among the different electron microscopy (EM) techniques available to visualize viral assembly at the intracellular level, we will focus on conventional ultrathin sections, cryosections, and freeze-substitution. For obtaining molecular information associated to ultrastructure we have now a group of methods to detect viral proteins (immunogold labeling), as well as the viral genome, through the different techniques for detection of nucleic acids (the enzyme-gold approach, in situ hybridization, and elemental mapping). We will illustrate the applications of these methods with examples of viruses that exhibit different levels of structural complexity. These new approaches help to detect and identify viruses in clinical samples and to characterize the virus life cycle and the cellular components involved, to obtain data that could help for a therapeutic intervention, and to characterize virus-like particles that can be the basis of new and safe vaccines.     

Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids

Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02–1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5′-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.     

Volumetric properties of nucleic acids

Volumetric studies can yield useful new information on a myriad of intra- and intermolecular interactions that stabilize nucleic acid structures. In particular, appropriately designed volumetric measurements can characterize the conformation-dependent hydration properties of nucleic acids as a function of solution conditions, including temperature, pressure, ionic strength, pH, and cosolvent concentration. We have started to accumulate a substantial database on volumetric properties of DNA and RNA, as well as on related low molecular weight model compounds. This database already has provided unique insights into the molecular origins of various nucleic acid recognition processes, including helix-to-coil and helix-to-helix conformational transitions, as well as drug-DNA interactions. In this article, we review recent progress in volumetric investigations of nucleic acids, emphasizing how these data can be used to gain insight into intra-and intermolecular interactions, including hydration properties. Throughout this review, we underscore the importance of volume and compressibility data for characterizing the hydration properties of nucleic acids and their constituents. We also describe how such volumetric data can be interpreted at the molecular level to yield a better understanding of the role that hydration can play in modulating the stability and recognition of nucleic acids.     

A comparison of nucleic acid amplification techniques for the assessment of bacterial viability

AIMS: The ability to determine the presence and viability status of bacteria by molecular methods could offer significant advantages to the food, environmental and health sectors, in terms of improved speed and sensitivity of detection. METHODS AND RESULTS: In this study, we have assessed three amplification techniques, PCR, RT-PCR and NASBA, for their ability to detect nucleic acid persistence in an E. coli strain following heat-killing. NASBA offered the greatest sensitivity of the three methods tested. The presence of residual DNA and mRNA could be detected by PCR and NASBA, respectively, for up to 30 h postdeath, by which time cell death had been confirmed by culture methods. Thus a single quantitative measurement based on nucleic acid amplification did not permit unequivocal determination of cell viability. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation between cell viability and persistence of nucleic acids must be well characterized for a particular analytical situation before molecular techniques can be substituted for traditional culture methods.     

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.     

Single-molecule spectroscopic methods

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms.