CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza.

The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity with minimal equipment requirements are urgently needed to detect the many circulating species, subtypes, and variants of influenza at the point of need. Here, we introduce such a method using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a clustered regularly interspaced short palindromic repeats (CRISPR)-based RNA detection platform. Four SHINE assays were designed and validated for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously. This study demonstrates the utility of this duplex assay in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as in simultaneously detecting influenza A and human RNAse P in patient samples. These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance.

Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants.

The widespread transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for rapid nucleic acid diagnostics that are easy to use outside of centralized clinical laboratories. Here we report the development and performance benchmarking of Cas13-based nucleic acid assays leveraging lyophilised reagents and fast sample inactivation at ambient temperature. The assays, which we named SHINEv.2 (for 'streamlined highlighting of infections to navigate epidemics, version 2'), simplify the previously reported RNA-extraction-free SHINEv.1 technology by eliminating heating steps and the need for cold storage of the reagents. SHINEv.2 detected SARS-CoV-2 in nasopharyngeal samples with 90.5% sensitivity and 100% specificity (benchmarked against the reverse transcription quantitative polymerase chain reaction) in less than 90 min, using lateral-flow technology and incubation in a heat block at 37 °C. SHINEv.2 also allows for the visual discrimination of the Alpha, Beta, Gamma, Delta and Omicron SARS-CoV-2 variants, and can be run without performance losses by using body heat. Accurate, easy-to-use and equipment-free nucleic acid assays could facilitate wider testing for SARS-CoV-2 and other pathogens in point-of-care and at-home settings.

Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.

Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2.

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (SHERLOCK and HUDSON Integration to Navigate Epidemics), a sensitive and specific integrated diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We combine the steps of SHERLOCK into a single-step reaction and optimize HUDSON to accelerate viral inactivation in nasopharyngeal swabs and saliva. SHINE's results can be visualized with an in-tube fluorescent readout - reducing contamination risk as amplification reaction tubes remain sealed - and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-PCR with a sample-to-answer time of 50 minutes. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.

Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2.

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE's results can be visualized with an in-tube fluorescent readout - reducing contamination risk as amplification reaction tubes remain sealed - and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.