Analytical performance, sample preparation, and validation of CRISPR-Cas biosensors for point-of-care deployment

초록

CRISPR-Cas biosensors have moved from genome-editing tools toward analytical platforms for nucleic acid detection. Despite rapid technical progress, their analytical performance under realistic conditions remains uneven. This review critically examines CRISPR-Cas biosensing from an analytical chemistry standpoint, focusing on signal generation, assay kinetics, sample preparation, quantitative readout, and validation in complex matrices. We summarize how target recognition translates into measurable signals through trans-cleavage by Cas12, Cas13, and Cas14 and through Cas3-mediated cis-cleavage, and we discuss how guide RNA design, mismatch tolerance, and reaction kinetics determine analytical sensitivity and selectivity. Amplification-coupled assays built on RPA, LAMP, and NASBA are compared with amplification-free approaches in terms of reported LOD, dynamic range, and susceptibility to nonspecific signal. Fluorescence, colorimetric, lateral-flow, electrochemical, and luminescent readouts are evaluated against the requirements of quantitation, calibration, and reproducibility, with attention to one-pot and closed-tube workflows that constrain contamination. Sample preparation and on-chip integration are reviewed not only as engineering choices but as analytical variables that govern recovery, inhibitor carryover, and matrix bias. We then compare reported analytical metrics of CRISPRCas biosensors with those of established nucleic acid testing methods and identify recurring validation gaps such as small clinical cohorts, limited inter-laboratory data, and inconsistent reference materials. The review is intended to help analytical chemists distinguish CRISPR assay formats that are analytically mature from those that remain proof-of-concept, and to guide the design of future studies aimed at potentially field-compatible diagnostics.

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