SCOPE: Flexible targeting and stringent CARF activation enables type III CRISPR-Cas diagnostics

Summary Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA (rather than DNA) and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of a target RNA and production of cyclic oligoadenylate (cOA) second messenger molecules that may trigger dormancy or cell death. In this study, we discovered that a largely exposed seed region at the 3’ end of the crRNA is essential for target RNA binding and cleavage, whereas base pairing at a unique region at the 5’ end of the guide is required to trigger cOA production. Moreover, we uncovered that the natural variation in the composition of type III complexes within a single host results in different guide lengths, and hence variable seed regions. This shifting seed may prevent escape by invading genetic elements, while controlling cOA production very tightly to prevent unnecessary damage to the host. Lastly, we used these findings to develop a new diagnostic tool, named SCOPE, which was used for the specific detection of SARS-CoV-2 from human nasal swab samples, showing sensitivities in the atto-molar range.