Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9

Microfluidic vortex shedding (mu VS) can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state. The mechanistic underpinning of mu VS intracellular delivery remains undefined and mu VS-Cas9 genome editing requires further studies. Herein, we evaluated a series of mu 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 mu VS 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 (e mu VS). mu 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 e mu VS with negligible impact on cell viability. Herein, we demonstrate efficient processing of 5x10(6) cells suspend in 100 mu l of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 10(6) to 10(8) in 1 to 30 s. Cumulatively, these results demonstrate the rapid and robust utility of mu VS and e mu VS for genome editing human primary T cells with Cas9 RNPs.

Automated summary

The study evaluated a series of microfluidic vortex shedding (mu VS) devices with splitter plates, selected a design for efficient knockout of the endogenous T cell receptor in human T cells using Cas9 ribonucleoprotein (RNP) delivery, and demonstrated a significant increase in editing efficiency with e mu VS while minimizing impact on cell viability. This demonstrates the rapid and robust utility of mu VS and e mu VS for genome editing human primary T cells with Cas9 RNPs.