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

Bryce D Shirk1, Cecilia Z Rodriguez2, Marisa O Pacheco2

  • 1J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, 32611.

Biochemical Engineering Journal
|February 6, 2026
PubMed
Summary
This summary is machine-generated.

This study establishes CRISPR/Cas9 homology-directed repair (HDR) in insect cells for precise genome editing. Optimizing conditions and cell cycle synchronization significantly improved HDR efficiency for non-viral gene delivery.

Keywords:
CRISPR/Cas9Homology-Directed Repair (HDR)Insect cellcell synchronizationgenome editinghydroxyurea

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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.