Exploiting\nthe Potential of Spherical PAM Antenna\nfor Enhanced CRISPR-Cas12a: A Paradigm Shift toward a Universal Amplification-Free\nNucleic Acid Test Platform

The CRISPR-Cas12a system has shown\ntremendous potential\nfor developing\nefficient biosensors. Albeit important, current CRISPR-Cas system-based\ndiagnostic technologies (CRISPR-DX) highly rely on an additional preamplification\nprocedure to obtain high sensitivity, inevitably leading to issues\nsuch as complicated assay workflow, cross-contamination, etc. Herein,\na spherical protospacer-adjacent motif (PAM)-antenna-enhanced CRISPR-Cas12a\nsystem is fabricated for universal amplification-free nucleic acid\ndetection with a detection limit of subfemtomolar. Meanwhile, the\nclinical detection capability of this sensor was further verified\nusing gold-standard real-time quantitative polymerase chain reaction\nthrough Mycobacterium tuberculosis measurement,\nwhich demonstrated its good reliability for practical applications.\nImportantly, its excellent sensitivity is mainly ascribed to high\nefficiency of target search induced by a localized PAM-enriched microenvironment\nand improved catalytic activity of Cas12a (up to 4 folds). Our strategy\nprovides some new insights for rapid and sensitive detection of nucleic\nacids in an amplification-free fashion.