Genome editing human primary T cells with microfluidic vortex shedding & CRISPR Cas9

Microfluidic vortex shedding ( μVS ) can rapidly deliver mRNA to T cells with high yield. The mechanistic underpinning of μVS intracellular delivery remains undefined and μVS -Cas9 genome editing requires further studies. Herein, we evaluated a series of μVS devices containing splitter plates to attenuate vortex shedding and understand the contribution of computed force and frequency on efficiency and viability. We then selected a μVS design to knockout the expression of the endogenous T cell receptor in primary human T cells via delivery of CRISPR-Cas9 ribonucleoprotein (RNP) with and without brief exposure to an electric field ( eμVS ). μVS alone resulted in an equivalent yield of genome-edited T cells relative to electroporation with improved cell quality. A 1.8-fold increase in editing efficiency was demonstrated with eμVS with negligible impact on cell viability. Cumulatively, these results demonstrate the utility of μVS and eμVS for genome editing human primary T cells with Cas9 RNPs. ### Competing Interest Statement All authors except G.T.S.K. are consultants, employees, shareholders and/or optionees of Indee. Inc. or the wholly-owned Australian subsidiary Indee. Pty. Ltd. Both legal entities have an interest in commercializing microfluidic vortex shedding (PCT/AU2015/050748, PCT/AU2018/051190) and related technologies. G.T.S.K. declares no competing interest.