Enhancing CRISPR homology directed repair in IAL-PiD2 insect cells via reagent delivery optimization and cell synchronization

CRISPR/Cas9-mediated homology-directed repair (HDR) enables precise genome editing, yet its application in insect cell lines remains largely unexplored. Insect systems, already used in recombinant protein production via baculovirus expression, offer substantial potential for stable, non-viral genetic modification using HDR-based approaches. This study establishes a robust, reproducible HDR framework in Plodia interpunctella IAL-PiD2 cells, a lepidopteran species increasingly relevant in pest management and biomaterials research. We systematically evaluated factors influencing HDR efficiency, including transfection reagent, Cas9:sgRNA molar ratios, donor DNA concentration, homology arm length, and cell cycle synchronization. A ribonucleoprotein molar ratio of 1:1 and 0.66 pmol donor DNA template (∼3.97 × 10 ¹¹ copies) yielded the highest integration efficiency under standard transfection conditions. Cell cycle synchronization using hydroxyurea followed by transfection 4 h post-treatment produced a 1.57-fold increase in HDR efficiency versus asynchronous controls. This is the first demonstration of regulated cell cycle timing improving HDR in insect cells, emphasizing the importance of delivery timing for efficient editing. These findings establish cost-effective, scalable protocols for non-viral gene delivery in insect cells and position IAL-PiD2 as a viable platform for functional genomics, precision pest control, and recombinant protein production. The results also support in vivo applications of HDR, enabling precise genetic modifications for a broad range of investigative and applied purposes.