Microfluidic vortex shedding (µVS) can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state. 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 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. Herein, we demonstrate efficient processing of 5 × 106 cells suspend in 100 µl of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 106 to 108 in 1 to 30 s. Cumulatively, these results demonstrate the rapid and robust utility of µVS and eµVS for genome editing human primary T cells with Cas9 RNPs.

Microfluidic vortex shedding ( µVS ) can rapidly deliver mRNA to human T cells with high yield and minimal perturbation. However, the mechanistic underpinning of µVS as an intracellular delivery method remains undefined with no optimization framework. Herein, we evaluated a series of µVS devices containing various splitter plates to attenuate vortex shedding and understand the contribution of force and frequency on expression efficiency and cell viability. We selected and applied a µVS design to knockout the expression of the endogenous T cell receptor of T cells via delivery of Cas9-RNP. 255 µVS samples were characterized across more than 150 parameters and machine learning was used to identify the 11 most predictive parameters for expression efficiency and cell viability. T hese results demonstrate the utility of µVS for genome editing of human T cells with CRISPR-Cas9 and provide a robust framework to optimize µVS for various constructs, cell types and protocols.This work was funded in part by IndieBio (indiebio.co), SOSV (sosv.com), Jobs for NSW