Defining the allosteric role of RNA in the CRISPR-associated Cas13a protein activation

Cas13a is one of the latest additions in the CRISPR-Cas library that exclusively targets RNA, a remarkable property being leveraged for the development of tools for RNA detection, regulation, and imaging. Cas13a uses a CRISPR RNA (crRNA) sequence as a guide to target RNA sequences, which are cleaved through two Higher Eukaryotes and Prokaryotes Nucleotide (HEPN) catalytic domains. It has been demonstrated that the binding of a target RNA activates the spatially distant catalytic HEPN domains through allosteric regulation that has not been clarified. Here, extensive molecular simulations are combined with biochemical experiments to unravel the molecular basis of the RNA-mediated allosteric regulation in Cas13a. We reveal that the signal transduction pathway is activated by the target RNA and mediated by the interactions of the guide crRNA at the interface residues of HEPN domains. Perturbation in such interaction pattern, either in the presence of an extended crRNA-target RNA complementarity or in the complete absence of the target RNA, inactivate the catalytic cleft, proposing a molecular switch for Cas13a function. This characterization of the dynamic requirements underlying the RNA-mediated allosteric mechanism in Cas13a poses a fundamental understanding that can be instrumental in the development of advanced RNA-based editing and detection tools.