Integrating CRISPR/Cas Biosensors with Advanced Platforms: A Holistic Path Toward Preamplification-free, Multiplexed, and Continuous Molecular Monitoring.

Amplification-free CRISPR/Cas biosensors for point-of-care nucleic acid detection: recent advances and future perspectives.

Nucleic acid biomarkers are critical targets for early diagnosis of disease, public health surveillance and environmental safety. However, their low abundance and the complexity of the sample matrix pose strict requirements for the high sensitivity and portability of detection technologies. Although traditional clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems exhibit advantages such as high specificity, operational simplicity, and compatibility with mild reaction conditions, their reliance on pre-amplification steps elevates the risk of false-positive results and hinders their broader application. To overcome these limitations, amplification-free CRISPR/Cas technologies have emerged and undergone extensive development. These approaches enable highly sensitive nucleic acid detection without the need for pre-amplification and are more amenable to integration with portable devices, thereby offering promising avenues for point-of-care testing (POCT). This review systematically examines the fundamental principles and design strategies underlying amplification-free CRISPR/Cas biosensors and summarizes recent advances in detection platforms based on autocatalytic signal enhancement, nanomaterial-coupled amplification, and integrated high-sensitivity readout systems, while also outlining their practical applications in POCT settings. Furthermore, the key technical challenges and future development directions of amplification-free CRISPR technologies are discussed based on current advances in the field. These insights and perspectives aim to provide a systematic reference for further research and to facilitate the expanded application of amplification-free CRISPR/Cas systems in POCT.

CRISPR/cas-based biosensors for point-of-care testing: a comprehensive review of signal readout strategies

Evolution of CRISPR-cas detection systems: From nucleic acid recognition to omnidirectional point-of-care diagnostic applications.

Molecular diagnostic technologies play an indispensable role in modern medicine and public health. However, traditional diagnostic platforms frequently face an inherent trade-off between laboratory-grade analytical precision and the speed and operational simplicity required for point-of-care testing. In recent years, the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein system has precipitated disruptive technological changes to this field. CRISPR-Cas system possesses high-fidelity target recognition capability and exhibits a distinctive trans-cleavage activity upon activation, which functions as signal amplification. This technology alleviates the inherent trade-off between sensitivity and portability. This review systematically summarizes the core molecular mechanisms of CRISPR-Cas detection platforms, addressing the differences in substrate preference and cleavage behavior among mainstream effector proteins (e.g., Cas9, Cas12, Cas13, and Cas14) and prokaryotic Argonaute (pAgo) proteins. Furthermore,this review sorts out the technological iteration path of detection platforms and presents the applications of this technology in fields such as infectious disease surveillance, cancer liquid biopsy, preliminary screening of genetic diseases, food and environmental safety, and veterinary port quarantine. Despite the challenges in quantitative accuracy and anti-interference ability, CRISPR biosensors are powerfully driving precision medicine towards decentralized, on-site, and accessible Point-of-Care Testing (POCT).

Advances in cascaded CRISPR for preamplification-free nucleic acid assays.

The development of rapid, sensitive, and specific nucleic acid assays is pivotal for advancing molecular detection in clinical diagnosis, food safety, and environmental monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems, renowned for their programmable and signal amplification capacity, have emerged as efficient tools for meeting these challenges. However, the intrinsic sensitivity of CRISPR/Cas assays relying on a single Cas effector is typically confined to the picomolar level, often necessitating complex nucleic acid preamplification. Cascaded CRISPR systems, which integrate sequential enzymatic reactions or multiple CRISPR effectors, can address this limitation by achieving nucleic acid preamplification-free signal enhancement. This review starts with the introduction of the design principles and working mechanisms of cascaded CRISPR strategies, encompassing Enzyme-Coupled cascades, Multi-effector class 2 CRISPR cascades, and Type III CRISPR-mediated cascades. Then, we highlight the deployment of these techniques across diverse bio-sensing scenarios, ranging from disease diagnosis to food and environmental surveillance. Finally, critical challenges and emerging frontiers are discussed, including integration with digital detection platforms and AI-assistant algorithms.

LAMP-CRISPR Integrated Platforms for Rapid Detection of Microbial Pathogens: Principles, Applied Strategies, and the Road to Field Translation.

Loop-mediated isothermal amplification (LAMP) integrated with CRISPR-Cas systems has emerged as a promising molecular diagnostic platform for the rapid detection of microbial pathogens. By combining the efficient nucleic acid amplification of LAMP with the sequence-specific recognition capability of CRISPR-Cas effectors, these platforms offer potential advantages in analytical sensitivity, specificity, operational simplicity, and field applicability. In this review, we summarize the principles, assay formats, and recent advances of LAMP-CRISPR technologies for detecting a broad spectrum of microbial pathogens, including bacterial, viral, fungal, and parasitic agents across clinical, veterinary, food safety, environmental, and agricultural applications. Representative studies are compared with attention to pathogen type, sample matrix, assay design, CRISPR-Cas system, readout format, analytical performance, and practical application. We further discuss major technical challenges that continue to hinder practical implementation, particularly complex sample pretreatment, workflow integration, carry-over contamination, reagent stability, multiplexing capability, and platform standardization. Attention is given to sample pretreatment and system-level integration, including current extraction and rapid lysis strategies, closed-tube reactions, portable readouts, and microfluidic or cartridge-based formats, which may support simplified "sample-in, answer-out" diagnostic workflows. Finally, we outline future directions for improving matrix-adapted sample processing, assay robustness, standardized validation, large-scale evaluation, and field deployment. This review provides a structured overview of current LAMP-CRISPR platforms and highlights key technological considerations for translating rapid microbial pathogen detection from laboratory research to real-world applications.

A low-cost CHA-integrated CRISPR/Cas12a-based test strip platform for on-site gene detection.

On-site nucleic acid detection plays a crucial role in disease diagnosis, biosafety monitoring, and food quality control. This study develops a novel nucleic acid detection platform that integrates catalytic hairpin assembly (CHA) with the CRISPR/Cas12a system and utilizes pregnancy test strips (PTS) for result visualization, addressing the limitations of existing nucleic acid detection methods in balancing sensitivity, specificity, and portability with cost and dependence on a cleanroom. The main mechanism involves the following three steps. The presence of target RNA triggers the CHA reaction, generating double-stranded DNA (dsDNA) as an activation unit. Subsequently, this unit activates the CRISPR/Cas12a system to specifically cleave the single-stranded DNA (ssDNA) that has bridged human chorionic gonadotropin (HCG) to a magnetic bead, ultimately releasing HCG that produces a visual result on the PTS. This dual-signal amplification strategy (CHA cycling and Cas12a trans-cleavage) can detect concentrations as low as 10 pM in approximately 50 min, without the need for pre-amplification of the target nucleic acid. This detection system ensures high sensitivity and specificity while effectively avoiding non-specific activation. In practical applications with transgenic maize samples, the detection results are highly consistent with those of real-time quantitative polymerase chain reaction (qPCR), validating its reliability in real-world scenarios. This innovative method offers advantages such as simple operation and low cost, providing an efficient tool for rapid nucleic acid detection while demonstrating broad potential for application in resource-limited settings.