Abstract

CRISPR-Cas13d cleaves RNA and is used in vivo and for diagnostics. However, a systematic understanding of its RNA binding and cleavage specificity is lacking. Here, we describe an RNA Chip-Hybridized Association-Mapping Platform (RNA-CHAMP) for measuring the binding affinity for > 10,000 RNAs containing structural perturbations and other alterations relative to the CRISPR RNA (crRNA). Deep profiling of Cas13d reveals that it does not require a protospacer flanking sequence but is exquisitely sensitive to secondary structure within the target RNA. Cas13d binding is penalized by mismatches in the distal crRNA-target RNA region, while alterations in the proximal region inhibit nuclease activity. A biophysical model built from these data reveals that target recognition initiates in the distal end of the target RNA. Using this model, we design crRNAs that can differentiate between SARS-CoV-2 variants by modulating nuclease activation. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme.