Abstract: Induced pluripotent stem cells (iPSCs) have become an essential research platform to study various human diseases and hold significant potential for clinical development. The CRISPR/Cas9 system represents another breakthrough in biomedical research, enabling precise genome editing in mammalian cells. In our current workflow, we utilized the Neon™ NxT Electroporation System with the 8-Channel Pipette for rapid optimization of electroporation parameters for editing iPSCs. This system is a powerful tool for streamlining the optimization of electroporation programs and conditions for primary cells and cell lines. We demonstrated the system's performance in efficiently optimizing CRISPR/Cas9-based non-viral genome editing of iPSCs. Using the Neon™ NxT 8-Channel Pipette, we screened different gene delivery and editing conditions in high throughput format and generated chimeric antigen receptor (CAR)-iPSCs by delivering CD38-targeted anti MESO3 donor DNA and CTS™ HiFi™ Cas9 to iPSCs. Editing efficiency and cell viability were measured using flow cytometry and Next Generation Sequencing. Our results highlight the capacity to simultaneously test multiple electroporation conditions, significantly reducing the time required for optimization experiments. Through systematic screening of voltage, pulse duration, and pulse number, we optimized the delivery of CRISPR/Cas9 ribonucleoprotein (RNP) complexes while maintaining high cell viability. Furthermore, we evaluated different electroporation buffers and achieved up to 70% CD38 knockout and up to 30% CAR-positive iPSCs. These findings emphasize the critical role of buffer components in transfection efficiency and cell viability. In summary, the Neon™ NxT Electroporation System has proven to be an effective tool for the rapid and efficient optimization of non-viral genome editing in iPSCs. This capability significantly advances iPSC-based research and therapeutic applications, addressing scalability issues and improving patient outcomes.
