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Background Cell-based therapies have untapped potential as treatments for a wide range of diseases, with chimeric antigen receptor (CAR) T cells emerging as a leading treatment option for various hematological cancers. Despite recent advancements, there are still challenges around the efficacy, safety, and scalability of CAR T cell therapies. Non-viral engineering may address these concerns, reducing immunogenicity and manufacturing costs while maintaining T cell viability and functionality. Several gene editing technologies have emerged for stable, non-viral expression of CARs in primary human T cells.Methods For these approaches to be successful, highly efficient delivery of biomolecules and genome editing tools is essential. To this end, we developed optimized cell engineering workflows using the ExPERT™ electroporation platform that enabled highly efficient delivery of transposons and CRISPR-Cas editing elements for CD19 CAR T cell manufacturing. In a second approach, these two technologies were combined in a multiplexed editing strategy to generate allogeneic CD33 CAR T cells (CD33-AlloCART) as a potential treatment for acute myeloid leukemia (AML).Results This study focused on maximizing transposition efficiency, TRAC knockout, or HDR template knock-in, while maintaining cell expansion, recovery, and functionality. MaxCyte ® electroporation also enabled the simultaneous delivery of two CRISPR RNPs targeting the TRAC and B2M loci, Sleeping Beauty 100X transposase (SB100X) mRNA and CD33-CAR transposon minicircle DNA to generate functional CD33-AlloCART cells at a clinically-relevant scale.Conclusions Together, these results demonstrate the capability of MaxCyte’s clinically validated ExPERT™ electroporation platform to enable non-viral engineering of CAR T cells using transposon or CRISPR/Cas systems.Acknowledgements Data generated by MaxCyte Technical Applications Lab or in collaboration with CIMA/Clinica Universidad de Navarra and CIEMAT. Adapted with permission.