Massively parallel profiling of RNA-targeting CRISPR-Cas13d

Hung-Che Kuo (), Joshua Prupes, Chia-Wei Chou and Ilya J. Finkelstein ()
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Hung-Che Kuo: University of Texas at Austin
Joshua Prupes: University of Texas at Austin
Chia-Wei Chou: University of Texas at Austin
Ilya J. Finkelstein: University of Texas at Austin

Nature Communications, 2024, vol. 15, issue 1, 1-13

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-44738-w

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