Selective RNA pseudouridinylation in situ by circular gRNAs in designer organelles

Abstract RNA modifications play a pivotal role in the regulation of RNA chemistry within cells. Several technologies have been developed with the goal of using RNA modifications to regulate cellular biochemistry selectively, but achieving selective and precise modifications remains a challenge. Here, we show that by using designer organelles, we can modify mRNA with pseudouridine in a highly selective and guide-RNA-dependent manner. We use designer organelles based on the principle of phase separation, a central tenet in developing artificial membraneless organelles in living mammalian cells. In addition, we used circular guide RNAs to markedly enhance the effectiveness of targeted pseudouridinylation. Our studies offer spatial engineering by means of optimized RNA editing organelles (OREO) as a complementary tool in using targeted RNA modification to expand potential avenues for future investigation.