A Digital Microfluidic Electroporation Platform for Low-input CRISPR Genome Editing and mRNA Transfection In...

Area of Science:

• Biotechnology
• Cellular Engineering
• Molecular Biology

Background:

• Digital microfluidics (DMF) allows for precise control over small fluid volumes.
• Electroporation is a key method for introducing molecules into cells.
• Current methods often require high cell numbers and can impact cell viability.

Purpose of the Study:

• To develop and validate a high-throughput DMF electroporation workflow for genetic manipulation.
• To demonstrate efficient CRISPR-mediated gene editing in primary human T cells.
• To showcase mRNA delivery into 3D cell spheroids using DMF.

Main Methods:

• Spatially depositing CRISPR guide RNAs and assembling ribonucleoprotein (RNP) complexes on-chip.
• Utilizing DMF for electroporation of primary human suspension T cells (CD4+ and CD8+).
• Adapting the DMF workflow for mRNA transfection of 3D HEK293T spheroids.

Main Results:

• Achieved efficient TRAC locus disruption in T cells using only 10,000 cells, with >85% viability.
• Demonstrated robust and uniform EGFP mRNA expression in HEK293T spheroids without affecting morphology.
• Identified polymer additives that stabilize Cas9-sgRNA complexes for reproducible editing.

Conclusions:

• DMF electroporation provides a scalable, low-input solution for genetic manipulation of diverse cell types.
• This technology is suitable for applications in CAR-T cell therapy, functional genomics, and 3D cell models.
• The platform enables efficient genome editing and mRNA delivery in both suspension cells and multicellular spheroids.