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CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. However, these methods generally require several hours and could cause amplification errors, due to the pre-amplification of target nucleic acids to enhance the detection sensitivity. Here, we developed a platform that allows “CRISPR-based amplification-free digital RNA detection (SATORI)”, by combining CRISPR-Cas13-based RNA detection and microchamber-array technologies. SATORI detected single-stranded RNA targets with maximal sensitivity of ~10 fM in <5 min, with high specificity. Furthermore, the simultaneous use of multiple different guide RNAs enhanced the sensitivity, thereby enabling the detection of the SARS-CoV-2 N-gene RNA at ~5 fM levels. Therefore, we hope SATORI will serve as a powerful class of accurate and rapid diagnostics.

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The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29, and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11–crRNA–Csx29 complex with and without target RNA, and demonstrate that target RNA-binding induces conformational changes in Csx29. Biochemical experiments revealed target RNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE, suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11–Csx29–Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11–Csx29 effector is an RNA-dependent nuclease-protease.