Selective RNA pseudouridinylation in situ by circular gRNAs in designer organelles

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 inspired by concepts of phase separation, a central tenet in developing artificial membraneless organelles in living mammalian cells. In addition, we use circular guide RNAs to markedly enhance the effectiveness of targeted pseudouridinylation. Our studies introduce spatial engineering through optimized RNA editing organelles (OREO) as a complementary tool for targeted RNA modification, providing new avenues to enhance RNA modification specificity.