Intersegmental transfers drive target search in an RNA-targeting CRISPR system

Sequence-specific RNA-binding proteins (RBPs) must efficiently locate their targets among a multitude of cellular RNAs. Cas13, an RNA-guided CRISPR protein, represents an ideal model system in which to study this search process. Cas13 combats bacteriophage infection by cleaving RNA nonspecifically upon binding of its crRNA to the target RNA sequence; thus, Cas13s search for its RNA target comes with a time constraint determined by the rate of phage multiplication. The mechanism by which Cas13 locates its target within this critical window remains unknown. Here, we investigate Cas13s mechanism of target search through integration of biophysical modeling, activity assays, and biochemical characterization. We show that Cas13 employs facilitated diffusion to accelerate its search, and find that Cas13s search time when targeting RNAs of different lengths cannot be explained by 1D sliding, the search mechanism used by many DNA-binding proteins. We propose that Cas13 primarily searches for its RNA target by intersegmental transfers (ITs), non-specifically binding the RNA at two locations and directly switching between them without fully dissociating from the RNA. We develop a biophysical model for ITs in an RNA context that we subsequently validate experimentally. Furthermore, we demonstrate that ITs can differentially accelerate the search process for a broad class of RNA-binding proteins, as opposed to their DNA-binding counterparts, due to RNAs short persistence length and the heterogeneity of RNA lengths in the cell. Our results illuminate how Cas13 achieves rapid target recognition in a complex RNA environment, and implicate ITs as a potentially widespread solution to the RNA search problem.