Prime editor with rational design and AI-driven optimization for reverse editing window and enhanced fidelity.

Prime editing (PE) is a precise tool for introducing genetic mutations in eukaryotes. Extending the efficient editing scope and mitigating undesired byproducts are possible. We introduce reverse PE (rPE), a SpCas9-directed variant that enabled DNA editing at the 3' direction of HNH-mediated nick site. The rPE leveraging nCas9-D10A and rPE gRNA targeting the 5' direction of HNH-mediated nick site inscribes genetic alterations, achieving a reverse editing window and potentially high fidelity. HNH and reverse transcriptase engineered using protein language models in conjunction with La facilitate circular erPEmax and erPE7max, achieving editing efficiency up to 44.41% without nick gRNA or positive selection. Furthermore, our findings underscore the capability of rPE in inserting functionally enhanced variant (PIK3CD) for cell therapy. By expanding the editing scope and enhancing genomic manipulability, rPE represents a meaningful advancement in prime editing, improving its utility for research and therapeutic applications.