A Universal Proximity CRISPR Cas12a Assay for Ultrasensitive Detection of Nucleic Acids and Proteins

Herein, we describe a proximity CRISPR Cas12a assay that harnesses “collateral” single-stranded DNase activity of Cas12a as a universal amplifier for the ultrasensitive detection of nucleic acids and proteins. The target recognition is achieved through proximity binding rather than recognition by CRISPR RNA (crRNA), which allows the flexible assay design and expansion to proteins. A binding-induced primer extension reaction is then used to generate a predesigned CRISPR-targetable sequence as a barcode for signal amplification. We demonstrate that our assay is highly sensitive and universal. As low as 1 fM nucleic acid target could be detected isothermally in a homogeneous solution via the integration with nicking cleavage. We’ve also successfully adapted the assay for the sensitive and wash-free detection of antibodies in both buffer and diluted human serum samples.