Sensitive and multiplexed RNA detection with Cas13 droplets and kinetic barcoding (preprint)

Rapid and sensitive quantification of RNA is critical for detecting infectious diseases and identifying disease biomarkers. Recent direct detection assays based on CRISPR-Cas13a1-4 avoid reverse transcription and DNA amplification required of gold-standard PCR assays5, but these assays have not yet achieved the sensitivity of PCR and are not easily multiplexed to detect multiple viruses or variants. Here we show that Cas13a acting on single target RNAs loaded into droplets exhibits stochastic nuclease activity that can be used to enable sensitive, rapid, and multiplexed virus quantification. Using SARS-CoV-2 RNA as the target and combinations of CRISPR RNA (crRNA) that recognize different parts of the viral genome, we demonstrate that reactions confined to small volumes can rapidly achieve PCR-level sensitivity. By tracking nuclease activity within individual droplets over time, we find that Cas13a exhibits rich kinetic behavior that depends on both the target RNA and crRNA. We demonstrate that these kinetic signatures can be harnessed to differentiate between different human coronavirus species as well as SARS-CoV-2 variants within a single droplet. The combination of high sensitivity, short reaction times, and multiplexing makes this droplet-based Cas13a assay with kinetic barcoding a promising strategy for direct RNA identification and quantification.

Accelerated RNA detection using tandem CRISPR nucleases (preprint)

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.

Rapid detection of SARS-CoV-2 with Cas13 (preprint)

To combat disease outbreaks such as the COVID-19 pandemic, flexible diagnostics for rapid viral detection are greatly needed. We report a nucleic acid test that integrates distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We paired this workflow, termed Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER), with extraction-free sample lysis using shelf-stable reagents that are widely available at low cost. DISCoVER has been validated on saliva samples to incentivize frequent testing for more widespread community surveillance and robustly detected attomolar levels of SARS-CoV-2 within 30 minutes, while avoiding false positives in virus-negative saliva. Furthermore, DISCoVER is compatible with multiplexed CRISPR probes to enable simultaneous detection of a human gene control or alternative pathogens.