Ultrasensitive Detection of Nucleic Acid and Protein at Ambient Temperature by Using an Engineered CRISPR-Cas12a-Based Digital Assay

The development of sensitive and convenient molecular detection technologies is crucial for early disease diagnosis and precision medicine. The CRISPR-Cas12a system has garnered significant attention due to its efficient molecular detection capabilities. However, most methods were performed at 37 °C or higher, which require a temperature control system and limit their applicability in point-of-care (POC) settings. In this study, we demonstrate that LbCpf1-Ultra, an engineered CRISPR-Cas12a, exhibits robust trans-cleavage activity at room temperature. Leveraging this finding, we developed a versatile digital CRISPR platform capable of ultrasensitive detection of viral DNA and proteins at ambient temperature, achieving a limit of detection (LOD) of 11.9 copies/μL for DNA and 5 fM for proteins. To further enhance its usability in POC environments, we integrated this platform with a smartphone-based fluorescence imaging device, enabling low-cost and on-site detection of nucleic acids and proteins without the need for external equipment. Clinical validation showcases its potential for reliable diagnostics. This platform provides a new direction for future CRISPR-based molecular diagnostics and holds promise for clinical diagnosis and precision medicine.