Bulge DNA-driven CRISPR/Cas12a dynamic activation circuit enables highly sensitive and versatile biosensing.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are becoming powerful tools for disease biomarkers detection. Due to the specific recognition, cis-cleavage and nonspecific trans-cleavage capabilities, CRISPR/Cas systems have implemented the detection of nucleic acid targets (DNA and RNA) as well as non-nucleic acid targets (e.g., proteins, exosomes, cells, and small molecules). In this review, we first summarize the principles and characteristics of various CRISPR/Cas systems, including CRISPR/Cas9, Cas12, Cas13 and Cas14 systems. Then, various types of applications of CRISPR/Cas systems used in detecting nucleic and non-nucleic acid targets are introduced emphatically. Finally, the prospects and challenges of their applications in biosensing are discussed.

Isothermal nucleic acid amplification for food safety analysis

CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the "turn-off" signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a "turn-on" fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for "turn-on" fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a's trans-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a "turn-on" SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.

In fields such as the early diagnosis of diseases, food safety monitoring, and environmental pollutant screening, the requirements for the sensitivity, specificity, and efficiency of detection technologies are extremely stringent. Traditional detection techniques such as HPLC and ELISA are cumbersome, time-consuming, and costly, and cannot meet the needs of on-site rapid testing and trace target screening. Under this backdrop, the CRISPR-Cas system, with its high targeting specificity and programmability, has brought new directions to the development of biosensors, particularly promoting technological innovations in recognition mechanisms and signal amplification for the detection of non-nucleic acid targets. This study focuses on CRISPR-Cas system-based biosensors, analyzing their structural composition, comparing the detection approaches for nucleic acid and non-nucleic acid targets, and conducting a performance comparison with traditional technologies. Studies have shown that this sensor combines high sensitivity and specificity with low cost and rapid detection, is capable of detecting both nucleic acid and non-nucleic acid targets, and thus has broad application prospects. However, it still has some limitations, such as the complexity of the indirect recognition process and the constraints on signal amplification. The integration of signal amplification, AI, nanotechnology, and microfluidics in the future is expected to break through bottlenecks and achieve industrial applications.

Non-nucleic acid targets, consisting primarily of metal ions, organic small molecules and proteins. They act as important biomolecules or cell surface markers, supplying integrated and comprehensive bio-diagnostic information for the early diagnosis and treatment of diseases. Meanwhile, the analysis of non-nucleic acid targets also offers the foundation for individualized medicine and precision therapy. Therefore, a versatile platform for non-nucleic acid targets requires development. Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas) systems is driving a revolution in medical diagnostics due to high base-resolution and isothermal signal amplification. Nevertheless, the majority of CRISPR/Cas settings reported currently are targeted for nucleic acids, leaving restricted usage to non-nucleic acid targets. This is owing to the lack of suitable signal recognition transduction elements for connecting CRISPR to non-nucleic acid targets. Functional nucleic acids (FNAs), comprising aptamers and nucleic acid enzymes, are of great concern to the biological and medical professions because of their specific target recognition and catalytic properties. As appropriate, functional recognition elements, FNAs can be integrated into CRISPR/Cas systems to exploit the powerful capabilities of both. This review emphasizes the technical tricks of integrating CRISPR/Cas systems and FNAs for non-nucleic acid targeting diagnostic applications. We first offer a general overview and the current state of research in diagnostics for CRISPR/Cas and FNAs, respectively, highlighting strengths and shortcomings. A categorical summary of non-nucleic acid-targeted diagnostics is provided, with a key emphasis on fundamental insights into the versatile non-nucleic acid-targeted diagnostic toolbox. We then review emerging diagnostic strategies based on CRISPR/Cas systems and FNAs that are fast, accurate and efficient in detecting non-nucleic acid targets. Finally, we identify the challenges that remain in this emerging field and look to the future of the field, expanding to the integration of nanomaterials, development of wearable devices and point-of-care testing.

In addition to their roles as revolutionary genome engineering tools, CRISPR-Cas systems are also highly promising candidates in the construction of biosensing systems and diagnostic devices, which have attracted significant attention recently. However, the CRISPR-Cas system cannot be directly applied in the sensing of non-nucleic acid targets, and the needs of synthesizing and storing different vulnerable guide RNA for different targets also increase the application and storage costs of relevant biosensing systems, and therefore restrict their widespread applications. To tackle these barriers, in this work, a versatile CRISPR-Cas12a-based biosensing platform was developed through the introduction of an enzyme-free and robust DNA reaction network, the entropy-driven dynamic DNA network. By programming the sequences of the system, the entropy-driven catalysis-based dynamic DNA network can respond to different types of targets, such as nucleic acids or proteins, and then activate the CRISPR-Cas12a to generate amplified signals. As a proof of concept, both nucleic acid targets (a DNA target with random sequence, T, and an RNA target, microRNA-21 (miR-21)) and a non-nucleic acid target (a protein target, thrombin) were chosen as model analytes to address the feasibility of the designed sensing platform, with detection limits at the pM level for the nucleic acid analytes (7.4 pM for the DNA target T and 25.5 pM for miR-21) and 0.4 nM for thrombin. In addition, the detection of miR-21 or thrombin in human serum samples further demonstrated the applicability of the proposed biosensing platform in real sample analysis.

Nucleic acid nanotechnology has emerged as a transformative tool in tumor research due to several unique properties including exceptional programmability and biocompatibility. The simplicity of their synthesis and chemical modification, their versatility as probes for both nucleic acid and non-nucleic acid targets, and their compatibility with signal amplification strategies make nucleic acid nanostructures ideal for biosensing applications. To date, nucleic acid nanotechnology has been successfully used in the precise detection and monitoring of tumor biomarkers at multiple biological scales. Furthermore, the engineering of sensory and modulable nucleic acid nanostructures has facilitated breakthroughs at the single-cell level in illuminating and reprogramming the tumor microenvironment (TME), thereby advancing tumor diagnostics and therapeutic decision-making. Framework nucleic acids (FNAs) have also shown promise in immunomodulation, offering novel strategies for fine-tuning immune responses in cancer immunotherapy. This review highlights the role of nucleic acid nanotechnology in non-invasive imaging and biomarker profiling of the TME, with a focus on innovative approaches that enhance detection sensitivity and real-time monitoring. Furthermore, the advantages and potential applications of nucleic acid nanotechnology in cancer immunotherapy are discussed. Through a detailed exploration of these advances, this review aims to provide insights into the pivotal role of nucleic acid nanotechnology in deciphering and modulating the TME for enhanced therapeutic outcomes in oncology.

Novel rolling circle amplification biosensors for food-borne microorganism detection

DNAzyme motor systems using gold nanoparticles (AuNPs) as scaffolds are useful for biosensing and in situ amplification because these systems are free of protein enzymes, isothermal, homogeneous, and sensitive. However, detecting different targets using the available DNAzyme motor techniques requires redesigns of the DNAzyme motor. We report here a toehold-exchange translator and the translator-mediated DNAzyme motor systems, which enable sensitive responses to various nucleic acid targets using the same DNAzyme motor without requiring redesign. The translator is able to efficiently convert different nucleic acid targets into a specific output DNA that further activates the pre-silenced DNAzyme motor and consequently initiates the autonomous walking of the DNAzyme motor. Simply adjusting the target-binding region of the translator enables the same DNAzyme motor system to respond to various nucleic acid targets. The translator-mediated DNAzyme motor system is able to detect as low as 2.5 pM microRNA-10b and microRNA-21 under room temperature without the need of separation or washing. We further demonstrate the versatility of the translator and the DNAzyme motor by successful construction and operation of four logic gates, including OR, AND, NOR, and NAND logic gates. These logic gates use two microRNA targets as inputs and generate amplified fluorescence signals from the operation of the same DNAzyme motor. Incorporation of the toehold-exchange translator into the DNAzyme motor technology improves the biosensing applications of DNA motors to diverse nucleic acid targets.

The recently developed bio-barcode assay for the detection of nucleic acid and protein targets without PCR has been shown to be extraordinarily sensitive, showing high sensitivity for both nucleic acid and protein targets. Two types of particles are used in the assay: (i) a magnetic microparticle with recognition elements for the target of interest; and (ii) a gold nanoparticle (Au-NP) with a second recognition agent (which can form a sandwich around the target in conjunction with the magnetic particle) and hundreds of thiolated single-strand oligonucleotide barcodes. After reaction with the analyte, a magnetic field is used to localize and collect the sandwich structures, and a DTT solution at elevated temperature is used to release the barcode strands. The barcode strands can be identified on a microarray via scanometric detection or in situ if the barcodes carry with them a detectable marker. The recent modification to the original bio-barcode assay method, utilizing DTT, has streamlined and simplified probe preparation and greatly enhanced the quantitative capabilities of the assay. Here we report the detailed methods for performing the ligand exchange bio-barcode assay for both nucleic acid and protein detection. In total, reagent synthesis, probe preparation and detection require 4 d.

Self-phosphorylating DNAzyme DK1 enables programmable multi-analyte readout via PfAgo.

In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

A dual fragment triggered DNA ladder nanostructure based on logic gate and dispersion-to-localization catalytic hairpin assembly for efficient fluorescence assay of SARS-CoV-2 and H1N1

DNA-based probes constitute a versatile platform for making biological measurements due to their ability to recognize both nucleic acid and non-nucleic acid targets, ease of synthesis and chemical modification, amenability to be interfaced with signal amplification schemes, and inherent biocompatibility. Here, we provide a historical perspective of how a transition from linear DNA structures toward more structurally complex nanostructures has revolutionized live-cell analysis. Modulating the structure gives rise to probes that can enter cells without the aid of transfection reagents and can detect, track, and quantify analytes in live cells at the single-organelle, single-cell, tissue section, and whole organism levels. We delineate the advantages and disadvantages associated with different probe architectures and describe the advances enabled by these structures for elucidating fundamental biology as well as developing improved diagnostic and theranostic systems. We also discuss the outstanding challenges in the field and outline potential solutions.

The programmability and flexibility of the RNA-directed CRISPR/Cas12a system underpin its utility as a potent tool for diagnostic applications. However, existing engineered crRNA strategies are still limited by a narrow target range, inadequate specificity, and operational complexity. To overcome these challenges, a proximity-assembly and activate (PAA) strategy was developed, employing split dumbbell activators with terminally modified target-binding modules that reassemble on scaffold RNA to reconstruct functional crRNA and activate Cas12a trans-cleavage activity. The design allows universal detection of both nucleic acid and non-nucleic acid targets. Notably, owing to its strict target dependency, the assembled crRNA biosensor significantly reduces background signal and suppresses nonspecific leakage. We demonstrated that the PAA system facilitates rapid and highly specific detection of miRNA-21, ATP, and anti-Dig antibody in complex matrices, enabling single-base discrimination among miRNA variants. Moreover, the platform successfully detected endogenous miRNA-21 in serum and cellular samples from breast cancer patients, clearly distinguishing them from healthy controls. This work presents a modular, plug-and-play, and versatile platform for molecular diagnostics, holding considerable potential for advancing clinical diagnostics and precision medicine.