Electroporation-based CRISPR gene editing in adult buffalo fibroblast cells

Electroporation is a widely used method for delivering CRISPR components into cells; however, it presents challenges when applied to difficult-to-transfect cells like adult buffalo fibroblasts. In this study, the ITGB2 gene (encoding the CD18 protein), plays vital for cellular adhesion and immune responses, was selected for editing experiments. To optimize electroporation conditions, we investigated parameters such as electric field strength, pulse duration, plasmid DNA amount, cuvette type, and cell type. The best transfection rates were obtained in a 4 mm gap cuvette with a single 20-millisecond pulse of 300 V using a 10 mu g of all-in-one CRISPR plasmid for 106 cells in 100 mu L of electroporation buffer. Increasing DNA quantity enhanced transfection rates but compromised cell viability. The 4 mm cuvette gap had high transfection rates than the 2 mm gap, and newborn cells exhibited higher transfection rates than adult cells. We achieved transfection rates of 10-12% with a cell viability of 25-30% for adult fibroblast cells. Subsequently, successfully edited the ITGB2 gene with a 30% editing efficiency, confirmed through various analysis methods, including T7E1 assay, TIDE and ICE analysis, and TA cloning. In conclusion, electroporation conditions reported here can edit buffalo gene(s) for various biotechnological research applications.