CRISPR-Ultimate: Leveraging Thermostable Cas12b and Tnpb Enzymes for a PAM-Less, Extraction-Free Detection Platform

CRISPR based diagnostic platforms utilizing the collateral cleavage property of Cas12 and Cas13 enzymes have emerged as reliable tools for rapid, sensitive, and accurate nucleic acid detection. Given the potential for using these tools in point-of-care applications in resource-limited settings, there is a critical need to devise straightforward and efficient protocols for field use. Here we present CRISPR-ULTIMATE, a simple diagnostic platform based on the thermostable Cas12b and TnpB enzymes that can quickly detect nucleic acid targets from unextracted samples. Our platform combines nucleic acid extraction, RT-LAMP based amplification, and fluorescence readout in a single point reaction with a 5-minute heat inactivation step, thereby streamlining the assay preparation and reducing the risk of contamination. Importantly, along with a simplified assay, CRISPR ULTIMATE is also capable of detecting any target sequence without the constraint of a PAM or TAM sequence, thus making it a powerful tool for nucleic acid detection. We apply CRISPR-ULTIMATE for the direct detection of SARS-CoV-2 RNA from crude saliva samples providing a sample-to-result in less than 30 minutes. Thus, CRISPR-ULTIMATE promises to be a robust and powerful platform that can be used on the field or in a point-of-care setting.