SunTag-PE: a modular prime editing system enables versatile and efficient genome editing

• Published in: Communications biology Vol. 8; no. 1; pp. 452 - 9
• Main Authors: Liu, Jiashuo, Zhang, Jingjing, Zhao, Tingting, Zhao, Mengya, Su, Min, Chen, Ye, Huang, Zheying, Wang, Yuyan, Zhong, Chaoyue, Hu, Zheng, Zhou, Ping, Tian, Rui, He, Dan
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 18.03.2025; Nature Publishing Group; Nature Portfolio
• Subjects: 38/71; 631/1647/1511; 631/1647/2300/1992; Biomedical and Life Sciences; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems; Dependovirus - genetics; Efficiency; Gene deletion; Gene Editing - methods; Gene loci; Genetic disorders; Genetic transformation; Genome editing; Gynecology; HEK293 Cells; Hospitals; Humans; Insertion; Life Sciences; Modularity; Mutation; Obstetrics; Oncology; Packaging; Plasmids; Proteins; RNA-directed DNA polymerase
• ISSN: 2399-3642; 2399-3642
• DOI: 10.1038/s42003-025-07893-4

Prime editing (PE) holds tremendous potential in the treatment of genetic diseases because it can install any desired base substitution or local insertion/deletion. However, the full-length PE effector size (6.3-kb) is beyond the packaging capacity of adeno-associated virus (AAV), hindering its clinical translation. Various splitting strategies have been used to improve its delivery, but always accompanied by compromised PE efficiency. Here, we developed a modular and efficient SunTag-PE system that splits PE effectors into GCN4-nCas9 and single-chain variable fragment (scFv) tethered reverse transcriptase (RT). We observed that SunTag-PEs with 1×GCN4 in the N terminus of nCas9 was the most efficient configuration rather than multiple copies of GCN4. This SunTag-PE strategy achieved editing levels comparable to canonical fused-PE (nCas9 and RT are linked together) and higher than other split-PE strategies (including sPE and MS2-PE) in both PE2 and PE3 forms with no increase in insertion and deletion (indel) byproducts. Moreover, we successfully validated the modularity of SunTag-PE system in the Cas9 orthologs of SauCas9 and FrCas9. Finally, we employed dual AAVs to deliver SunTag-ePE3 and efficiently corrected the pathogenic mutation in HBB mutant cell line. Collectively, our SunTag-PE system provides an efficient modular splitting strategy for prime editing and further facilitate its transformation in clinics. This study presents a novel, modular prime-editing system, SunTag-PE, that achieves gene editing levels comparable to larger, fused systems, but can be efficiently delivered into cells using adeno-associated viruses thanks to its small size.