(543g) Invited Talk: Improving CRISPR-Cas9 Homology Directed Repair in Ial-PiD2 Insect Cells from Plodia Interpunctella

While CRISPR/Cas9 HDR has been widely applied for both in vitro and in vivo modifications across diverse organisms ^1-4, integration efficiency remains a considerable constraint and, in some species, remains largely unexplored. This limitation necessitates laborious screening and selection processes to enrich the edited population, reducing the overall efficiency of the system and complicating the establishment of homogenous cell populations or germline mutant strains ^5-7. To address this challenge, investigators have explored various strategies to enhance HDR efficiency, including optimizing the delivery method of the mutagenic reagents, refining design parameters and reagent concentrations ^8-10, and modulating intrinsic cellular factors such as synchronizing cell cycles to phases where HDR is most active ^{3, 11}. Many of these optimizations have been conducted in mammalian systems, where biological pathways and regulatory mechanisms may not be directly applicable to other organisms. Therefore, expanding these studies to encompass a broader range of species is necessary to fully capture the complexity of biological diversity. In this work, we transition these same investigations into an insect cell line derived from Plodia interpunctella Hübner (Lepidoptera: Pyralidae) imaginal disc, IAL-PiD2 ¹².

Plodia interpunctella has a well-documented history in pest management as a significant global pest that affects stored commodities and processed food products ¹³. More recently, it has emerged as a candidate for silk-based biomaterial production ^14-16. To validate and optimize CRISPR/Cas9 HDR in this system, the fibroin-heavy chain (Fib-H) gene was selected as the initial target ¹⁷. Fib-H is the primary structural component of Lepidopteran silk and a commercially significant biopolymer. While its amino acid composition and structure vary across species, it remains the foundation of silk fiber formation and is the key protein used in silk-based biomaterials. Most silk-based biomaterials¹⁸ are derived from fibroin proteins extracted from the domesticated silkworm, Bombyx mori; however, sourcing or modifying fibroins from alternative species could expand the range of material properties, offering new possibilities for medical and commercial applications.

In this study, several factors influencing CRISPR/Cas9 HDR knock-in efficiency were examined. First, the ratio of Cas9 to sgRNA plays a critical role in the formation of the ribonucleoprotein (RNP) complex, directly affecting target site recognition and cleavage efficiency. Additionally, the amount of donor DNA template can impact the availability of homologous sequences for repair, influencing integration success. To further enhance HDR, cell cycle synchronization was enhanced using 2-hydroxyurea, which arrests cells in the G0/G1 phase, allowing for delivery of the CRISPR/cas9 and sgRNA when homology-directed repair is most active. By optimizing these parameters, we improved targeted integration efficiency in P. interpunctella, IAL-PiD2, cells. These efforts are a critical step for using the IAL-PID2 cell line for future therapeutic protein production.

Figure 1 shows the general modification plan for the homology directed repair in the Fib-H gene, example images of modified IAL-PID2 cells expressing mNeonGreen, and the comparisons between cas9 and sgRNA concentrations for two homology arm lengths, supporting these results.

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