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Updated: May 16, 2025

Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
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Plasmid-based electroporation for efficient genetic engineering in immortalized T lymphocytes.

Yu-Qing Xie1, Martin Fussenegger2

  • 1Department of Biosystems Science and Engineering, Eidgenössiche Technische Hochschule Zurich, Schanzenstrasse 48, CH-4056, Basel, Switzerland.

Metabolic Engineering
|April 4, 2025
PubMed
Summary

We developed an optimized, cost-effective electroporation method for genetically engineering T-cells using plasmid DNA. This technique enhances T-cell research and preclinical studies for novel T-cell therapies.

Keywords:
CAR-TCRISPR/Cas9CTLL-2Cell engineeringElectroporationGenome editingHT-2ImmunotherapyMolecular cloningSynthetic biologyT-cells

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biotechnology

Background:

  • Clinical success of T-cell therapies necessitates improved genetic engineering methods.
  • Current viral transduction methods are labor-intensive and require strict biosafety.
  • Efficient, cost-effective T-cell genetic engineering tools are limited.

Purpose of the Study:

  • To establish a facile and cost-effective method for T-cell genetic engineering.
  • To optimize plasmid-based electroporation for immortalized T-cell lines.
  • To enable efficient gene delivery, editing, and integration in T-cells.

Main Methods:

  • Utilized immortalized murine T-cell lines (HT-2, CTLL-2) and Jurkat cells.
  • Optimized cuvette-based electroporation for high-efficiency transfection.
  • Employed plasmid constructs for large gene delivery (up to 6.5 kbp) and Sleeping Beauty transposon system for genomic integration.
  • Developed CRISPR/Cas9 methods for gene knockout and homology-directed repair (HDR) knock-in.

Main Results:

  • Achieved high transfection efficiencies in HT-2, CTLL-2, and Jurkat cells.
  • Successfully delivered large gene cargos and achieved stable genomic integration.
  • Demonstrated high gene knockout (up to 97%) and HDR knock-in (up to 70%) efficiencies using CRISPR/Cas9.
  • Validated HT-2 and CTLL-2 cell lines for recapitulating primary T-cell characteristics.

Conclusions:

  • Optimized plasmid-based electroporation provides a practical, cost-effective tool for T-cell genetic engineering.
  • This method accelerates fundamental research in lymphocyte biology.
  • Facilitates preclinical studies for advanced T-cell therapies.