Numerical optimization of microfluidic vortex shedding for genome editing human primary T cells using machine learning
Jarrell, J. A.; Lievano, A. A.; Pan, F. L.; Lau, K. H. W. J.; Kirby, G. T. S.; Pawell, R. S.
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Microfluidic vortex shedding (VS) can rapidly deliver mRNA to T cells with high yield. The mechanistic underpinning of VS intracellular delivery remains undefined and VS-Cas9 genome editing requires further studies. Herein, we evaluated a series of VS 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 VS 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 (eVS). VS 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 eVS with negligible impact on cell viability. Cumulatively, these results demonstrate the utility of VS and eVS for genome editing human primary T cells with Cas9 RNPs.
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