High-efficiency, site-specific integration of kilobase-scale DNA into plant genomic safe harbors via PrimeStack editors

Precise, site-specific integration of large DNA sequences into plant genomes is a cornerstone of crop biotechnology and synthetic biology, yet remains constrained by random insertion, inefficient homologous recombination, and gene targeting. Here, we present PrimeStack, a DSB-independent platform that integrates prime editing with the unidirectional large serine integrase Bxb1, leveraging evolved variants for enhanced activity, to achieve the programmable insertion of multigene, multikilobase cargos at predefined genomic safe harbors in rice. Optimized prime editors first install attP landing sites with high fidelity and heritability followed by Bxb1-mediated recombination that generates irreversible integration of genetic information. PrimeStack achieves integration frequencies of approximately 43–46% (as detected by junction-specific PCR in rice calli), with phenotypic neutrality in regenerated plants, comparing favorably with bidirectional Cre-lox systems. We validate its utility by achieving targeted insertion of a carotenoid cassette at an experimentally confirmed genomic safe harbor. PrimeStack delivers a modular, site-specific gene-stacking platform that enables predictable, multigene trait pyramiding and pathway construction at genomic safe harbors, thereby accelerating the development of improved and resilient crop varieties, as well as scalable plant-based biomanufacturing and a powerful chassis for synthetic biology.