Programmable kinetic barcoding for multiplexed RNA detection with Cas13a

Rapid identification of viral infections and specific variants in patient samples requires a simple and multiplexed RNA detection method that does not rely on DNA sequencing. Although recent direct detection assays based on CRISPR-Cas13a offer rapid RNA detection by avoiding reverse transcription and DNA amplification required of gold-standard PCR assays, these assays are not easily multiplexed to detect multiple viruses or variants without dividing the sample into separate reactions. Here we show that Cas13a acting on single-target RNAs exhibits variable nuclease activity that depends on the interaction between the target RNA and crRNA. To exploit this feature for multiplexed detection, we devised a crRNA modification strategy that enables programmable tuning of Cas13a's nuclease enzymatic rates. Using a droplet-based Cas13a assay, we demonstrate that kinetic signatures can be harnessed to differentiate among respiratory viruses and SARS-CoV-2 variants in contrived and clinical samples. This kinetic barcoding strategy can be extended to additional RNA targets through simple modification of crRNAs.