Phase-Separated RNA Condensates Govern Cas13 Target Accessibility and Cleavage

Abstract RNA-guided CRISPR-Cas13 nucleases must efficiently locate and target specific transcripts amidst the millions of other RNA molecules that are spatially regulated in the cell. Yet the mechanisms by which Cas13 finds its targets within this crowded, compartmentalized environment remain elusive. Here, we show that diverse Cas13 orthologs assemble into distinct cytoplasmic granules in a crRNA-concentration dependent manner. These granules exhibit hallmark features of phase-separated condensates, displaying liquid-like viscoelasticity and dynamic molecular exchange with the cytoplasm, as demonstrated by FRAP analysis in both human and bacterial cells. Molecular profiling revealed that Cas13 co-localizes with polyadenylated RNAs and noncoding RNAs within condensates that display markers of canonical RNA granules. Biochemical purification coupled with RNA sequencing shows Cas13 associated with thousands of transcripts within condensates, likely mediated by electrostatic interactions with its positively charged surface. Notably, Cas13 retains catalytic activity within these condensates, efficiently cleaving co-localized targets, whereas RNA species excluded from the condensates remain largely protected from Cas13 cleavage. This data indicates that condensate-based spatial organization facilitates efficient sampling and binding of diverse RNA targets by concentrating Cas13 and its substrates within a confined, liquid-like compartment. Together, our findings uncover a conserved spatial mechanism regulating Cas13 activity across bacterial and mammalian cells, where localization within RNA dense granules governs Cas13 activity in cells.