PrimeStack editors enable efficient and site-specific integration of large multigene cargos in plants
- 1Laboratory for Genome Engineering and Synthetic Biology, Division of Biomedical Sciences, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia.
Abstract
Precise, site-specific integration of large DNA sequences into plant genomes is a long-standing goal 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 PE 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 (GSH) in rice (Oryza sativa). 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 enabled targeted large-cargo integration in regenerated rice plants with efficiencies of up to 45%, as determined by junction-specific PCR analyses. No obvious morphological differences from WT plants were observed at 5 weeks after transfer to soil under the evaluated conditions. 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 GSH, thereby accelerating the development of improved and resilient crop varieties, as well as scalable plant-based biomanufacturing and a powerful chassis for synthetic biology.
Precise integration of large DNA sequences into plant genomes remains a major challenge. This study presents PrimeStack, a platform that combines prime editing with the Bxb1 integrase to achieve the programmable insertion of multigene cargos at plant genomic safe harbors with high efficiency.
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BibTeX
@article{sanchez2026primestack,
title = {{PrimeStack editors enable efficient and site-specific integration of large multigene cargos in plants}},
author = {Edith Sánchez and Khalid Sedeek and Haroon Butt and Magdy Mahfouz},
journal = {PLOS Biology},
year = {2026},
month = oct,
doi = {10.1371/journal.pbio.3003778},
url = {https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003778},
note = {CC BY},
}
RIS
TY - JOUR
TI - PrimeStack editors enable efficient and site-specific integration of large multigene cargos in plants
AU - Edith Sánchez
AU - Khalid Sedeek
AU - Haroon Butt
AU - Magdy Mahfouz
PY - 2026
DA - 2026/10/13
JO - PLOS Biology
DO - 10.1371/journal.pbio.3003778
UR - https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003778
AB - Precise, site-specific integration of large DNA sequences into plant genomes is a long-standing goal 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 PE 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 (GSH) in rice (Oryza sativa). 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 enabled targeted large-cargo integration in regenerated rice plants with efficiencies of up to 45%, as determined by junction-specific PCR analyses. No obvious morphological differences from WT plants were observed at 5 weeks after transfer to soil under the evaluated conditions. 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 GSH, thereby accelerating the development of improved and resilient crop varieties, as well as scalable plant-based biomanufacturing and a powerful chassis for synthetic biology. Precise integration of large DNA sequences into plant genomes remains a major challenge. This study presents PrimeStack, a platform that combines prime editing with the Bxb1 integrase to achieve the programmable insertion of multigene cargos at plant genomic safe harbors with high efficiency.
SN - 1545-7885
SN - 1544-9173
ER -