Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

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Robust, sensitive, and specific diagnostic platforms for early pathogen detection are essential for the identification of infected patients and management of current and future pandemics. CRISPR-Cas systems have been repurposed for SARS-CoV-2 detection in two-pot assays. Two-pot assays require extra steps and are prone to cross-contamination; however, the temperature range of current Cas enzymes limits the development of one-pot assays Here, we characterized TccCas13a, a thermophilic Cas13a enzyme with cis and trans activities from 37–70°C, and HheCas13a, which had a limited range and lower activity. We harnessed TccCas13a in a one-pot SARS-CoV-2 assay with two layers of amplification and TccCas13a-mediated collateral degradation of a single-stranded RNA reporter. This assay showed 95% sensitivity and 100% specificity compared with RT-qPCR on clinical samples. We also developed a mobile phone application to facilitate data reading, collection, and sharing. Our OPTIMA-dx detection module exhibits key features for point-of-care SARS-CoV-2 screening and pathogen detection in general.