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

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 (6.3kb) was beyond the packaging capacity of adeno-associated virus (AAV), hindering its clinical transformation. 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 one 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 and higher than other split PE strategies (including sPE and MS2 PE) in both PE2 and PE3 forms with no increase in insertion/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. ### Competing Interest Statement The authors have declared no competing interest.