A High-Throughput Microdroplet-Based Single Cell Transfection Method for Gene Knockout Based on the CRISPR/Cas9...

Area of Science:

• Biotechnology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 gene editing requires efficient intracellular delivery of reagents.
• Traditional methods like lipotransfection have low efficiency, high toxicity, and are cell-type dependent.

Purpose of the Study:

• To develop a high-throughput microdroplet-based method for single-cell transfection.
• To improve the efficiency and accuracy of delivering genome-editing reagents into diverse cell types.
• To optimize DNA transfection conditions for specific cell lines using the microdroplet method.

Main Methods:

• A high-throughput, quantitative DNA transfection method using microdroplets for single-cell delivery.
• Exploration of optimal DNA concentrations and transfection conditions for various cell lines.
• Utilizing a single-cell microfluidic chip for CRISPR/Cas9 DNA transfection.

Main Results:

• Achieved high-efficiency delivery of nucleic acids into different single cell types by controlling plasmid numbers in microdroplets.
• Optimized transfection conditions led to successful construction of gene-knockout cancer cell lines.
• Demonstrated inhibition of migration ability and reduced tumorigenicity in TRIM72 knockout cancer cells in a zebrafish model.

Conclusions:

• The microdroplet-based method significantly enhances CRISPR/Cas9 gene-editing efficiency, especially in difficult-to-transfect cells.
• This technique offers a powerful alternative for precise intracellular gene delivery in biological research and therapeutic development.