CRISPR RNA Engineering Enables Single Nucleotide Polymorphism Discrimination in Nucleic Acid Detection.

Thermodynamically programmed one-pot CRISPR platform for point-of-care SNP genotyping

One-pot CRISPR diagnostics face a fundamental incompatibility: isothermal nucleic acid amplification enables rapid target accumulation, whereas CRISPR activation irreversibly consumes those substrates, destabilizing reaction kinetics. Here we show that reaction order can be programmed into DNA primers through thermodynamic design. Differences in primer-binding strength create two sequential amplification stages, delaying CRISPR activation until enough amplicons have accumulated without physical separation or external control. The design also introduces the protospacer adjacent motif (PAM), a short sequence required for CRISPR recognition, through the primer rather than relying on its presence in the native target, expanding target accessibility while retaining single-nucleotide discrimination. An ordinary differential equation model captures the threshold behavior and establishes a predictable framework for primer design. Building on this principle, we develop Thermodynamically Encoded Molecular Programming for One-pot diagnostics (TEMPO), which achieves attomolar sensitivity within 30 min and enables sequencing-concordant SNP genotyping and pathogen detection in a single-step microfluidic format. One-pot CRISPR diagnostics are limited by kinetic conflict between isothermal amplification and CRISPR detection. By thermodynamically programming reaction order into DNA primers, the authors create staged amplification enabling rapid, sensitive, single-step nucleic acid testing.

Strategic Design and Engineering of CRISPR/Cas-Powered Sensing Platforms for Enhanced Nucleic Acid Detection.

Rapid and accurate nucleic acid detection is fundamental to effective disease management. While PCR remains the gold standard, its requirement for sophisticated instrumentation limits its application in point-of-care settings. CRISPR-Cas systems have emerged as a disruptive diagnostic technology, leveraging the programmable specificity and unique trans-cleavage activity of Cas effectors to revolutionize biosensing. This review systematically evaluates the evolution of CRISPR-Cas-powered sensing platforms, categorized by their signal transduction modalities. We first discuss the expanding biochemical landscape of Cas nucleases, highlighting recent discoveries where conventional boundaries of Cas9, Cas12, and Cas13 have been transcended to enable versatile DNA/RNA targeting. Subsequently, we provide a comprehensive analysis of four primary sensing architectures: (1) Fluorescence-based platforms, exploring diverse strategies from target and signal amplification with dual-labeled ssDNA probes to nanomaterial-based probes; (2) Naked-eye visual platforms, encompassing both solid-phase lateral flow assays and solution-phase colorimetric strategies that facilitate rapid, instrument-free screening; (3) Electrochemical biosensors, which transduce biological recognition events into measurable electrical parameters, offering high sensitivity and seamless integration with miniaturized electronics; and (4) Electronic and Optoelectronic systems, including field-effect transistors and plasmonic sensors, which offer high-sensitivity, label-free detection. Despite significant progress, the translation of CRISPR-Dx from laboratory proof of concepts to clinical reality faces several bottlenecks. We critically analyze current challenges, including the need for integrated "sample-to-answer" workflows, high-throughput multiplexing, and digital quantification. Finally, we envision future trends such as AI-assisted signal processing and wearable sensing interfaces. By bridging the gap between molecular biology and advanced engineering, CRISPR-powered platforms are poised to make precision molecular diagnostics universally accessible.

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).

Engineering DNA-Targeting CRISPR and CRISPR-Like Effectors: Advances from Rational Design and High-Throughput Screening to AI-Driven Development

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute adaptive immune systems in prokaryotes and have transformed life sciences, precision medicine, and synthetic biology as programmable genome-editing tools. Despite their broad utility, naturally occurring DNA-targeting Cas effectors remain constrained by several intrinsic limitations, including large protein size that complicates delivery, stringent protospacer adjacent motif (PAM) requirements that restrict targetable genomic space, and mismatch tolerance that can lead to off-target activity and potential genotoxicity. These challenges have made Cas protein engineering and the discovery of novel CRISPR and CRISPR-like systems from metagenomic resources central to the development of next-generation genome-editing platforms. This Review places recent advances within an integrated synthetic biology engineering continuum that links natural effector discovery, structure-guided hypothesis generation, high-throughput functional screening, machine learning-enabled model construction, and iterative redesign. This Review summarizes progress in the screening, optimization, and functional engineering of DNA-targeting CRISPR and CRISPR-like effectors, with emphasis on structure-guided rational design, directed evolution coupled with high-throughput screening, bioinformatics- and evolution-guided mining of novel systems from large-scale sequence databases, and artificial intelligence-assisted development. By integrating these strategies, we highlight how CRISPR effector engineering is moving toward design-build-test-learn (DBTL)-inspired workflows that expand the functional landscape of genome-editing technologies and advance genome editing toward improved efficiency, safety, and programmability.

Cas12a/crRNA Recognition Catalyzed Triple Self-Priming Cascade Amplification Strategy for Sensitive and Reliable Drug Resistance Gene Analysis.

Chemical additives-enhanced CRISPR/Cas12a-based RNA detection.

The CRISPR/Cas12a system has revolutionized molecular diagnostics due to its RNA-guided trans-cleavage activity, enabling programmable and highly accurate nucleic acid detection. However, most Cas12a-based assays are optimized for DNA targets, while direct RNA detection constrained by limited sensitivity, typically at the nanomolar level. Existing strategies to improve the performance of RNA analysis often rely on additional DNA activators or complex auxiliary systems. Here, we report a simple yet effective chemical additive-based strategy that overcomes these limitations. This chemical additives-enhanced CRISPR/Cas12a-based RNA detection (CARD) enables femtomolar-level RNA detection using only a single crRNA, without the need for DNA activators, reverse transcription, or strand-displacement reactions. Notably, this approach can be adapted to single-stranded DNA, enabling ssDNA detection at attomolar levels. Collectively, CARD provides a straightforward, amplification-free, and highly sensitive diagnostic framework that might be readily extended to other CRISPR/Cas systems for ultrasensitive nucleic acid diagnostics.