This work systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment.
Abstract
CRISPR-based molecular diagnostics have emerged as powerful and programmable platforms that enable sensitive and specific detection for disease management and epidemiological surveillance. Advances in CRISPR engineering and assay design are driving the emergence of next-generation detection platforms that are highly sensitive, rapid, and amenable to field deployment. These engineering breakthroughs have the potential to reshape point-of-care tests (POCT) and transform how emerging and persistent health threats are monitored in decentralized and resource-limited settings. Herein, we systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment. The discussed strategies encompass both the rational engineering of CRISPR ribonucleoproteins (RNPs) and the optimization of downstream signaling modules for molecular diagnostic applications. We further highlight key challenges and future perspectives that may inspire impactful research directions and accelerate the advancement of CRISPR engineering strategies toward robust, field-deployable POCT platforms.
A comprehensive evaluation of current applications demonstrates the efficacy of CRISPR-Cas12b-based diagnostics across diverse pathogens, including viruses, bacteria, and parasites, with a specific focus on its integration with isothermal amplification techniques.
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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 are introduced, including integration with digital detection platforms and AI-assistant algorithms are discussed.
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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 effector...
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system began as a tool for programmable genome editing. CRISPR/Cas technologies have evolved into a versatile platform for functional genomic screening, epigenome editing, therapeutic target discovery, and highly sens...
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Conventional molecular diagnostics, particularly polymerase chain reaction (PCR), remain the gold standard for pathogen detection but are often unsuitable for rapid point‐of‐care (POC) testing because they require specialized equipment and laboratory infrastructure. CRISPR‐Cas systems have emerged as powerful alterna...