PrimeStack 编辑器可实现植物中大型多基因货物的高效和特定位点整合
来源 PLOS Biology 开放期刊23 天前
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作者:Edith Sánchez、Khalid Sedeek、Haroon Butt、Magdy Mahfouz 将大型 DNA 序列精确、位点特异性地整合到植物基因组中是一个长期目标...
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<p>by Edith Sánchez, Khalid Sedeek, Haroon Butt, Magdy Mahfouz</p>
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 (<i>Oryza sativa</i>). Optimized prime editors first install <i>attP</i> 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.
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