4.7 Article

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

Journal

SCIENTIFIC REPORTS
Volume 11, Issue 1, Pages -

Publisher

NATURE RESEARCH
DOI: 10.1038/s41598-021-91307-y

Keywords

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Funding

  1. IndieBio
  2. SOSV
  3. Jobs for NSW
  4. Y Combinator
  5. AusIndustry
  6. Social Capital
  7. Main Sequence Ventures
  8. Founders Fund's FF Science
  9. Axial
  10. MTP Connect Biomedtech Horizons 1.0
  11. NSW Health Medical Device Fund
  12. National Cancer Institute [75N91020C00030]

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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.
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.

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