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Background CAR T cell therapy has had a tremendous impact on the treatment of hematological cancers. However, these cell products could be improved through the addition or removal of factors that may enhance persistence, trafficking, and cytotoxicity, to name a few. Portal is investigating both RNA-based and genome editing technologies (e.g. CRISPR/Cas9) to design cellular therapies (T and NK cell) with customized functionalities. One of the key advantages of this technology is its minimal disruption of gene expression while enabling efficient delivery of a wide range of cargoes to different cell types. This enables sophisticated cell engineering that can augment existing products or pursuit of novel therapeutics.Methods Primary immune cells were collected from healthy donors and mechanoporated using Portal’s gentle silicon membrane chips that contain pores sized according to the cell type of interest. Delivery and the functional outcome of this delivery was measured by flowcytometry.Results At Portal, we have successfully delivered CRISPR/Cas9 RNPs, mRNAs, circular RNAs, and siRNAs into T cells, B cells, NK cells, and monocytes. For instance, we achieved over 85% GFP expression and B2M deletion in naive T cells after simultaneous delivery of mRNA and CRISPR-RNPs. In activated T cells, delivery of circular RNAs led to expression of functional cargoes like membrane-bound IL-2 and chimeric antigen receptors (CARs) for as many as 7 days and maintained a signature of sustained signaling. Using a clinical-scale prototype, we have achieved delivery of over 1x10 9 T cells per minute, resulting in more than 50% knockout efficiency and 90% GFP expression after 7 days of T cell expansion.Conclusions Multiplexed engineering of T cells can significantly improve the safety and efficacy of immune cell therapies. This work demonstrates CRISPR/Cas9 RNPs, mRNAs, circular RNAs, and siRNAs can be successfully delivered as single cargos or in a multiplexed fashion, demonstrating the potential for either single- or multi-step cell engineering workflows which can address several targets. Circular RNA delivery of membrane bound IL-2 and IL-12 further illustrates the potential for greater clinical impact while eliminating the risks of genetic modification.