Potent and Durable Gene Silencing by Circularized Trans-Pistol Ribozymes

RNA-cleaving ribozymes hold considerable potential as a gene silencing platform. Despite continuous efforts over the past decades, their efficacy has been severely limited by insufficient cellular activity and poor structural stability. Here we present a potent and durable gene silencing tool constructed by circularizing trans-acting Pistol ribozymes. This rationally engineered ribozyme mediates efficient gene knockdown by directly cleaving target RNA transcripts, displaying broad applicability for various RNA species across different cell lines. Its circular architecture confers enhanced structural stability with sustained gene repression, while its intrinsic catalytic mechanism allows uncompromised multiplex gene knockdown. Mechanistic studies reveal that these ribozyme-cleaved RNA fragments persist within cells, which is a distinct outcome compared to conventional RNA degradation-based approaches. Utilization of this tool to silence key regulatory genes achieves efficient manipulation of cell responses and fates, delivering potent and durable antitumor activity without detectable side effects and immune responses in a mouse model. Our work establishes this circularized trans-cleaving ribozyme as a robust gene silencing tool and a promising RNA therapeutic candidate, laying a foundational framework for the development of diversified ribozyme-based gene manipulation techniques.