Enhancing CRISPR Homology Directed Repair in IAL-PiD2 Insect Cells via Reagent Delivery Optimization and Cell...

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9-mediated homology-directed repair (HDR) allows precise genome editing but is underutilized in insect cell lines.
• Insect cell systems are valuable for recombinant protein production and offer potential for stable, non-viral genetic modification via HDR.
• The lepidopteran cell line *Plodia interpunctella* IAL-PiD2 is relevant for pest management and biomaterials research.

Purpose of the Study:

• To establish a robust and reproducible HDR framework in *Plodia interpunctella* IAL-PiD2 insect cells.
• To systematically evaluate factors influencing HDR efficiency in this insect cell line.
• To demonstrate the utility of optimized HDR for functional genomics and applied purposes in insects.

Main Methods:

• Systematic evaluation of transfection reagents, Cas9:sgRNA molar ratios, donor DNA concentration, and homology arm length.
• Optimization of ribonucleoprotein (RNP) complex formation and donor DNA template amounts.
• Implementation of cell cycle synchronization using hydroxyurea to enhance HDR efficiency.

Main Results:

• A Cas9:sgRNA ribonucleoprotein molar ratio of 1:1 and 0.66 pmol of donor DNA template maximized integration efficiency.
• Cell cycle synchronization 4 hours post-hydroxyurea treatment increased HDR efficiency by 1.57-fold compared to asynchronous cells.
• This study presents the first evidence of cell cycle regulation enhancing HDR in insect cells.

Conclusions:

• Established cost-effective, scalable protocols for non-viral gene delivery in insect cells using HDR.
• Positioned IAL-PiD2 cells as a versatile platform for functional genomics, precision pest control, and recombinant protein production.
• Validated the potential for *in vivo* applications of HDR for precise genetic modifications in insects.