Expanding the cell quantity of CRISPR/Cas9 gene editing by continuous microfluidic electroporation chip.

• A LaViE-Chip is developed to genetically edit cells by CRISPR/Cas9 with high speed. • Uniform electrical field and hydrodynamic cell rotation are realized in LaViE-Chip. • 71.06 % electroporation efficiency and 84.3 % cell viability are achieved. CRISPR/Cas9-mediated gene editing offers promising and safe therapeutic options for a wide range of diseases. The technical difficulty of efficiently acquiring large quantities of gene-edited therapeutic cells in a short time period is now preventing the widespread clinical application of CRISPR/Cas9-mediated gene editing. Herein, a Large Volume Continuous Electroporation Chip (LaViE-Chip) has been developed to address the challenge of acquiring sufficient quantities of genetically edited cells for CRISPR/Cas9 gene editing. By connecting multiple relatively narrow microfluidic channels in parallel, a satisfactory balance between cell flow volume and electric field uniformity was achieved with two simple off-chip electrodes, which also isolated harmful effects around electrodes from target cells. Meanwhile, by carefully designing the curvature of the microfluidic channel, hydrodynamic controlled rotation of target cells has been realized to improve the transfection efficiency and cell viability. With these improvements, the LaViE-Chip realized 71.06 % electrotransfection efficiency, 84.3 % cell viability, and 107 cell/min cell processing speed. Moreover, the first successful incessant CRISPR gene editing by electroporation has been demonstrated, laying the technical foundation of therapeutic CRISPR gene editing. [ABSTRACT FROM AUTHOR]