Authors:

Syed S. Ali

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The advent of CRISPR/Cas9 gene-editing has many applications in basic science and precision medicine. Current methods rely on nucleofection to deliver CRISPR/Cas9 ribonucleoprotei n (RNP) complexes, which requires harsh electroporation to create pores in the plasma membra ne needed to internalize RNP complexes. Therefore, increasing the efficacy of gene editing wh ile maintaining the viability and function of the edited cell population is of the utmost import ance for designing gene therapies. As an alternative to existing nucleofection-based method s, we investigated the efficacy of delivering gene edits using a microfluidics-based system to int roduce a transient plasma membrane disruption (TPMD), allowing for the delivery of RNP co mplexes to T cells. To accomplish this, we first identified the optimal pressure for deli vering a fluorescent, FITC-Dextran cargo to resting fresh T cells as 70 PSI, with stark decrease s in cell viability at higher PSIs and frozen T cell populations. Following our initial optimi zation, we confirmed that following the delivery of a CRISPR/Cas9 RNP at a 6:1 sgRNA :Cas9 molar ratio to knockout (KO) the gene CD226, cells having undergone TPMD demonstrated comparable editing efficiency to nucleofection when evaluating CD226 expression, 4 days post-transfection using spectral flow cytometry. Lastly, we identified when stimul ating T cells immediately following TPMD-mediated editing, those cells demonstrate improved viability and proliferative capacity, measured by proliferation tracking dye dilution, comp ared to T cells having undergone nucleofection. Overall, our data demonstrate that TPMD-ba sed T cell gene-editing exhibits potential as an alternative to nucleofection-based methods. Fu ture experiments can be directed towards examining editing efficacy with greater cel l throughputs and determining the most optimal waiting period following TPMD, after which r esting T cells can be stimulated to achieve maximal proliferation for cell therapy applications.

Source:

University of Florida / Syed S. Ali, Rafi Rammoo, Pedro Zanoelo, Alexander D. Pearce, Matthew E. / 2024

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Co-authors:

Syed S. Ali