CRISPR-associated transposon for programmable viral vector engineering and prime editing.

Baculovirus, an insect virus commonly used for recombinant protein expression in insect cells and gene delivery in mammalian systems, is often generated through bacmid-based engineering. To enable flexible and programmable bacmid engineering, we developed SHOT 2.0, an optimized CRISPR-associated transposon platform that mediates RNA-guided and customized bacmid editing in Escherichia coli. The edited bacmid can be transfected into insect cells to produce recombinant baculoviruses. SHOT 2.0 supported site-specific integration of large DNA cargos (at least 14 kb) into defined loci such as v-cath and ODVe56, with integration at ODVe56 markedly improving transgene stability during serial virus passaging. The system is fully compatible with the Bac-to-Bac® workflow, enabling dual-gene insertion into the bacmid and derived baculovirus. Leveraging this platform, we constructed an all-in-one baculovirus encoding the PE5max prime editor. This vector-mediated prime editing achieves efficiencies up to 85.6% in HEK293T cells and achieves robust prime editing in hard-to-transfect cell types, including iPSCs and liver cancer cells, with efficiencies up to 37.1%. These results demonstrate that SHOT 2.0 substantially expands the baculovirus engineering toolbox, providing a flexible platform for genome editing and future gene delivery.