调控CRISPR-Cas9系统用于基因编辑的研究进展

基于操作简便、成本低、快捷高效的优势,规律间隔成簇短回文重复序列及其相关核酸酶(CRISPR-Cas9)系统在基因编辑的基础研究和临床医学方面起到了极为重要的推动作用.在时空维度上调控CRISPR-Cas9发挥功能,对于减少CRISPR-Cas9系统的脱靶效应,提高基因编辑的特异性具有重要意义.本综述介绍了近年来利用化学分子以及光调控CRISPR-Cas9系统发挥功能,进而调控基因编辑的研究进展,并探讨了CRISPR-Cas9调控的前景和面临的挑战.     

Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.     

Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10–20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.     

DNA Origami Post-Processing by CRISPR-Cas12a

Customizable nanostructures built through the DNA-origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting-edge tools for DNA-origami design and preparation, it remains challenging to separate structural components of an architecture built from—thus held together by—a continuous scaffold strand, which in turn limits the modularity and function of the DNA-origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA-origami structures. We target single-stranded (ss) regions of DNA-origami structures and remove them with CRISPR-Cas12a, a hyper-active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post-processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a-like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.     

CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2

The CPV-2 DNA extracted from dog's intestine was amplified using RPA and transcribed into an RNA copy. The RNA copy was incubated with Cas13a and crRNA, upon activation collateral cleavage activity of Cas13a cleaves all the target RNA and nonspecific RNA in the vicinity producing fluorescent signals within 30 min.     

CRISPR-Cas System for RNA Detection and Imaging

The system of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated endonucleases(Cas)have been widely used in gene editing,disease treatment,molecular diagnosis and chromosome imaging.On account of the programmable target recognition of CRISPR-Cas system and the specific targeting function toward RNA of type Ⅵ class Ⅱ Cas proteins,CRISPR-Cas system has been deployed as RNA recognition and detection tools,exhibiting promising application potentials in the field of RNA detection and imaging.In this review,we summarize the latest research progresses as well as development prospects of CRISPR-Cas system in RNA diagnosis and live cell RNA imaging.     

基因调控中的DNA结合蛋白

DNA结合蛋白通过与特定位点的DNA结合可进行基因功能的调控。根据蛋白质结晶学和分子生物学研究了蛋白质-DNA之间的相互作用。对几种DNA结合域,如“螺旋-转折-螺旋”、“亮氮酸拉链”、“锌指”进行了讨论。     

How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.     

不同结构的DNA激活序列对Cas12a反式切割活性的影响

CRISPR－Cas12a系统的反式切割活性在其识别特定的DNA激活序列后被激活,这不仅能实现特定DNA靶标的直接定量分析,同时也为构建针对多种生物标志物的体外传感体系带来了新的思路。然而,已有文献中所采用的双链DNA（dsDNA）和单链DNA（ssDNA）激活序列结构多种多样,缺乏全面、系统的设计指导原则。针对该问题,该文系统研究了不同结构的DNA激活序列对LbaCas12a反式切割活性的影响。通过对比研究,得出以下结论：（1）前间区序列邻近基序（PAM）位点有助于LbaCas12a更高效地靶向结合dsDNA激活序列和ssDNA激活序列;（2）PAM近端区域缺少序列片段会降低Cas12a-crRNA定位激活序列的效率;（3）删除PAM远端序列片段有利于增强LbaCas12a的反式切割活性;（4）由于省略了dsDNA解链过程,ssDNA激活序列在激活LbaCas12a的反式切割活性方面普遍比dsDNA激活序列产生的效果更好。根据这些发现,该文提出了一种LbaCas12a所青睐的高效激活序列结构,其激活的LbaCas2a反式酶切活性较采用含PAM位点的标准dsDNA激活序列高出3.7倍。研究结果为构建基于CRISPR-Cas12a的高效体外生物传感系统提供了重要支撑。     

Programmable DNA Nanoflowers for Biosensing,Bioimaging, and Therapeutics

DNA nanostructures have shown excellent prospects in biomedical applications owing to their unique sequence programmability, function designability, and biocompatibility. As a type of unique DNA–inorganic hybrid nanostructures, DNA nanoflowers (DNFs) have attracted considerable attention in the past few years. Precise design of the DNA sequence enables the functions of DNFs to be customized. Specifically, DNFs exhibit high physiological stability and more diverse properties by virtue of the incorporation of inorganic materials, which in turn have been applied in an assortment of biomedical fields. In this review, the design, synthesis, and biomedical applications of programmable DNFs are discussed. First, the background of DNA-based materials and the fundamentals of DNFs are briefly introduced. In the second part, two synthetic methods of DNFs are categorized as the rolling circle amplification and salt aging method, focusing on the formation mechanism of DNFs and differences between the synthetic methods. In the third part, the biomedical applications of DNFs functional materials are summarized, including biosensing, bioimaging, and therapeutics. Finally, the challenges and future opportunities of DNFs are discussed toward more widespread applications.     

微纳米粒子的形貌调控及其对药物/基因传递体系的影响

超分子组装体由于具有适合的微纳米尺寸、可控的结构和良好的生物相容性等特点,极大地促进了药物/基因传递体系的发展。拓扑结构(如形貌、尺寸)是影响药物/基因传递体系的重要因素,这方面研究正成为这一领域的研究热点。本文综述了调控组装体形貌的主要手段,包括聚合物链结构与组成、组装条件、外界刺激及聚合诱导的组装体形貌,初步探讨了微观形貌对药物/基因传递体系的影响,并对这一领域进行了展望。     

基于上转换发光共振能量转移的CRISPR/Cas12a生物传感系统用于HPV16 DNA双信号检测

传统的纯核结构的上转换纳米材料用于生物传感时, 存在表面猝灭效应或者因发光共振能量转移(LRET)效率不高导致灵敏度低等缺点, 对目标物的灵敏检测有一定的局限性. 本文通过多步高温共沉淀, 介导壳层外延成长法, 合成了NaYF₄∶Yb³⁺,Er³⁺(C-UCNPs)核层发光和NaYF₄@NaYF₄∶Yb³⁺,Er³⁺@NaYF₄(CSS-UCNPs)内壳层发光能量限域型上转换纳米材料, 并表征了材料的晶型、 形貌、 表面配体、 元素组成和发光共振能量转移效率. 结果表明, 该材料具有表面猝灭效应低、 发光共振转移效率较高的优势, 随后将其与成簇规律间隔的短回文重复序列及其相关蛋白(CRISPR/Cas)12a-纳米金系统结合, 实现了人乳头瘤病毒DNA(HPV16 DNA)的比色定性和上转换发光定量分析, 检出限为69.8 pmol/L, 双信号检测有效提高了检测结果的准确性. 此外, 本方法不仅特异性强, 还能识别单碱基错配的HPV16 DNA, 提高了DNA片段的容错率.     

Functional Carbon Quantum Dots: A Versatile Platform for Chemosensing and Biosensing

Carbon quantum dot has emerged as a new promising fluorescent nanomaterial due to its excellent optical properties, outstanding biocompatibility and accessible fabrication methods, and has shown huge application perspective in a variety of areas, especially in chemosensing and biosensing applications. In this personal account, we give a brief overview of carbon quantum dots from its origin and preparation methods, present some advance on fluorescence origin of carbon quantum dots, and focus on development of chemosensors and biosensors based on functional carbon quantum dots. Comprehensive advances on functional carbon quantum dots as a versatile platform for sensing from our group are included and summarized as well as some typical examples from the other groups. The biosensing applications of functional carbon quantum dots are highlighted from selective assays of enzyme activity to fluorescent identification of cancer cells and bacteria.     

Transmembrane Signaling with Lipid‐Bilayer Assemblies as a Platform for Channel‐Based Biosensing

Artificial and natural lipid membranes that elicit transmembrane signaling is are useful as a platform for channel‐based biosensing. In this account we summarize our research on the design of transmembrane signaling associated with lipid bilayer membranes containing nanopore‐forming compounds. Channel‐forming compounds, such as receptor ion‐channels, channel‐forming peptides and synthetic channels, are embedded in planar and spherical bilayer lipid membranes to develop highly sensitive and selective biosensing methods for a variety of analytes. The membrane‐bound receptor approach is useful for introducing receptor sites on both planar and spherical bilayer lipid membranes. Natural receptors in biomembranes are also used for designing of biosensing methods.     

Whole-Genome Analysis of Acinetobacter baumannii Strain AB43 Containing a Type I-Fb CRISPR-Cas System: Insights into the Relationship with Drug Resistance

The CRISPR-Cas system is a bacterial and archaea adaptive immune system and is a newly recognized mechanism for controlling antibiotic resistance gene transfer. Acinetobacter baumannii (A. baumannii) is an important organism responsible for a variety of nosocomial infections. A. baumannii infections have become problematic worldwide because of the resistance of A. baumannii to multiple antibiotics. Thus, it is clinically significant to explore the relationship between the CRISPR-Cas system and drug resistance in A. baumannii. This study aimed to analyze the genomic characteristics of the A. baumannii strain AB3 containing the type I-Fb CRISPR-Cas system, which was isolated from a tertiary care hospital in China, and to investigate the relationship between the CRISPR-Cas system and antibiotic resistance in this strain. The whole-genome sequencing (WGS) of the AB43 strain was performed using Illumina and PacBio sequencing. The complete genome of AB43 consisted of a 3,854,806 bp chromosome and a 104,309 bp plasmid. The specific characteristics of the CRISPR-Cas system in AB43 are described as follows: (1) The strain AB43 carries a complete type I-Fb CRISPR-Cas system; (2) Homology analysis confirmed that the cas genes in AB43 share high sequence similarity with the same subtype cas genes; (3) A total of 28 of 105 A. baumannii AB43 CRISPR spacers matched genes in the bacteriophage genome database and the plasmid database, implying that the CRISPR-Cas system in AB43 provides immunity against invasive bacteriophage and plasmids; (4) None of the CRISPR spacers in A. baumannii AB43 were matched with antimicrobial resistance genes in the NCBI database. In addition, we analyzed the presence of antibiotic resistance genes and insertion sequences in the AB43 strain and found that the number of antibiotic resistance genes was not lower than in the “no CRISPR-Cas system” strain. This study supports the idea that the CRISPR-Cas system may inhibit drug-resistance gene expression via endogenous gene regulation, except to the published mechanism that the CRISPR-Cas system efficiently limits the acquisition of antibiotic resistance genes that make bacteria sensitive to antibiotics.     

Synthesis,Characterization, and Directional Binding of Anisotropic Biohybrid Microparticles for Multiplexed Biosensing

Anisotropic microarchitectures with different physicochemical properties have been developed as advanced materials for challenging industrial and biomedical applications including switchable displays, multiplexed biosensors and bioassays, spatially‐controlled drug delivery systems, and tissue engineering scaffolds. In this study, anisotropic biohybrid microparticles (MPs) spatio‐selectively conjugated with two different antibodies (Abs) are first developed for fluorescence‐based, multiplexed sensing of biological molecules. Poly(acrylamide‐co‐acrylic acid) is chemically modified with maleimide‐ or acetylene groups to introduce different targeting biological moieties into each compartment of anisotropic MPs. Modified polymer solutions containing two different fluorescent dyes are separately used for electrohydrodynamic co‐jetting with side‐by‐side needle geometry. The anisotropic MPs are chemically stabilized by thermal imidization, followed by bioconjugation of two different sets of polyclonal Abs with two individual compartments via maleimide‐thiol coupling reaction and Huisgen 1,3‐dipolar cycloaddition. Finally, two compartments of the anisotropic biohybrid MPs are spatio‐selectively associated with the respective monoclonal Ab‐immobilized substrate in the presence of the antigen by sandwich‐type immunocomplex formation, resulting in their ordered orientation due to the spatio‐specific molecular interaction, as confirmed by confocal laser scanning microscopy. In conclusion, anisotropic biohybrid MPs capable of directional binding have great potential as a new fluorescence‐based multiplexing biosensing system.

     

Biocompatible Infinite‐Coordination‐Polymer Nanoparticle–Nucleic‐Acid Conjugates for Antisense Gene Regulation

Herein, we report the synthesis of DNA‐functionalized infinite‐coordination‐polymer (ICP) nanoparticles as biocompatible gene‐regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3‐hydroxy‐4‐pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe^III to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe^III–HOPO ICP particles by copper‐free click chemistry afforded colloidally stable nucleic‐acid nanoconstructs. The DNA–ICP particles, when cross‐linked through sequence‐specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA–ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.