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 RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. 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 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (C t ) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.

Cas12a (Cpf1) is a bacterial RNA-guided nuclease used widely for genome editing and diagnostic applications. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity using functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit double-stranded DNA (dsDNA) recognition with AcrVA4 driving Cas12a dimerization. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophage with tools to evade CRISPR-Cas12a and support biotechnological applications where multiple-turnover enzymatic inhibition of Cas12a are desirable.

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

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.

In Fig. 3d, a spurious black color key appeared above the red dotted line for detection accuracy; this has now been removed.In the Results section, second paragraph, eighth sentence under heading "Detecting viral RNA with FIND-IT on a compact detector, " the sentence now reading "Based on the C t values of all COVID-19-positive samples (n = 39) identified in samples from the IGI testing laboratory (n = 308), " shows the correct number of samples from the IGI testing laboratory (308 vs. "296" as previously written).In the Acknowledgements section, grant R33AI140465 was missing and has been added to the sentence now reading "the work was also supported by the Howard Hughes Medical Institute (HHMI) and the National Institutes of Health (NIH) (R01GM131073 and DP5OD021369 to P.D.H., R01GM127463 to D.F.S., and 5R61AI140465-03 and R33AI140465 to J.A.D., D.A.F. and M.O.). " Further in the Acknowledgements section, the following sentence has now been included: "The computational aspects of this work related to SARS-CoV-2 guide RNA design were supported by the AWS Diagnostic Development Initiative via computational credit. "The online version of the Article has been updated.