Prime editing-installed suppressor tRNAs for disease-agnostic genome editing

Article Prime editing-installed suppressor tRNAs for disease-agnostic genome editing https://doi.org/10.1038/s41586-025-09732-2 Received: 2 March 2025 Accepted: 9 October 2025 Published online: 19 November 2025 Open access Check for updates Sarah E. Pierce1,2,3,6, Steven Erwood1,2,3,6, Keyede Oye1,2,3, Meirui An1,2,3, Nicholas Krasnow1,2,3, Emily Zhang1,2,3, Aditya Raguram1,2,3, Davis Seelig4, Mark J. Osborn5 & David R. Liu1,2,3 ✉ Precise genome-editing technologies such as base editing1,2 and prime editing3 can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration4,5 or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay–Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases. Therapeutic genome-editing efforts, including more than 70 clinical trials so far, have predominantly used programmable nucleases, base editors or prime editors to disrupt or correct disease-associated genes in an allele-specific manner. These approaches have proven to be effective in patients or in animal models for the treatment of disorders such as sickle-cell disease6,7, T cell leukaemia8, hypercholesterolaemia9,10, alpha-1-antitrypsin deficiency10, chronic granulomatous disease11, progeria12, spinal muscular atrophy13, prion disease14, alternating hemiplaegia of childhood15 and many other genetic diseases. Although allele-specific therapeutic genome-editing strategies offer treatments for many serious diseases with few treatment options, the breadth of the global genetic disease crisis, in which more than 8,000 genetic diseases collectively affect hundreds of millions of patients, demands new approaches that more rapidly bring the benefits of therapeutic genome editing to large numbers of patients16. Allele-specific applications of nucleases, base editors and prime editors require the development of a distinct genome-editing treatment for each of the more than 200,000 known pathogenic mutations, although prime editing also enables corrections of clustered hotspots of mutations. Until substantial streamlining of regulatory, development and manufacturing costs occurs, the development of the thousands of distinct drugs needed to treat any large fraction of patients with genetic disease remains impractical, despite the fact that base editing and prime editing can collectively correct the vast majority of known pathogenic mutations1–3. The versatility of prime editing, which uses a programmable nickase and a reverse transcriptase to replace targeted segments of DNA with new sequences of our choosing3, can be used in creative ways that may benefit far larger cross-sections of patients with genetic disease. Nonsense mutations account for 24% of pathogenic alleles in the ClinVar database16. The common molecular consequence of nonsense mutations—termination of translation before functional protein can be made—suggests more generalized approaches to their therapeutic rescue that might be applied in an allele-agnostic or even disease-agnostic manner. Nonsense sup-tRNAs have an anticodon that complements a premature termination codon (PTC) and support the installation of an amino acid rather than termination of translation. Although sup-tRNAs can theoretically read through both PTCs and natural termination codons (NTCs), extensive evidence suggests that sup-tRNAs can be Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA. 2Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. 3Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. 4Department of Veterinary Clinical Sciences, University of Minnesota, Minneapolis, MN, USA. 5 Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN, USA. 6These authors contributed equally: Sarah E. Pierce, Steven Erwood. ✉e-mail: 1 Nature | Vol 648 | 4 December 2025 | 191 Article well-tolerated in eukaryotes. Both naturally occurring and exogenous sup-tRNAs can be expressed without apparent toxicity4,5. Multiple biological mechanisms explain the observation of surprisingly low levels of sup-tRNA-mediated readthrough at NTCs. First, the distribution of stop codons for PTCs is distinct from the distribution for NTCs, particularly increasing the safety profile of sup-tRNAs corresponding to the amber stop codon (TAG)17. Second, the frequent presence of redundant and diverse in-frame stop codons following NTCs reduces the likelihood of extending a protein past a suppressed stop codon by more than a few amino acids18. Third, recruitment of polypeptide chain release factors to the 3′ untranslated region (3′ UTR) near NTCs can outcompete sup-tRNAs19. Fourth, if the ribosome continues past the NTC, the RNA and protein are targeted for degradation through the non-stop decay pathway20, and proteins that are translated into the 3′ UTR are further recognized and targeted for degradation21. Finally, sup-tRNAs mediate nonsense suppression only in transcripts that are being expressed in a given cell, minimizing the risk of toxicity from ectopic overexpression. Despite the promise of sup-tRNAs as a potential therapeutic strategy, current approaches such as lipid nanoparticle (LNP)-based or adeno-associated virus (AAV)-mediated delivery of sup-tRNAs4,5 face the challenge of supporting sup-tRNA production throughout the lifetime of the patient. AAV-based methods, in particular, may be limited to a single dose, owing to the generation of high levels of neutralizing antibodies following dosing, which may not be sufficient to treat genetic diseases that require re (...truncated)