Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering
Raes, Laurens; Pille, Melissa; Harizaj, Aranit; Goetgeluk, Glenn; Van Hoeck, Jelter; Stremersch, Stephan; Fraire, Juan C.; Brans, Toon; de Jong, Olivier Gerrit; Maas-Bakker, Roel; Mastrobattista, Enrico; Vader, Pieter; De Smedt, Stefaan C.; Vandekerckhove, Bart; Raemdonck, Koen; Braeckmans, Kevin
(2021) Molecular Therapy - Nucleic Acids, volume 25, pp. 696 - 707
(Article)
Abstract
The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In
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the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.
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Keywords: CRISPR-Cas9, gene editing, intracellular delivery, photoporation, stem cells, T cells, Drug Discovery, Molecular Medicine, Journal Article
ISSN: 2162-2531
Publisher: Nature Publishing Group
Note: Funding Information: We acknowledge the Centre for Advanced Light Microscopy at Ghent University (Belgium) for use of its microscopes and support for the microscopy experiments. The authors would like to acknowledge funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreements number (no.) 810685 ( DelNam project ) and no. 648124 . S.S. acknowledges the support of a VLAIO grant (grant number: HBC.2017.0542 ). M.P. ( FWO grant G036727N and FWO-SB grant 1S14318N ), J.V.H. ( FWO-SB grant 1S62519N ), and J.C.F. ( FWO grant 1210120N ) thank the financial support by the Flemish Research Foundation . Funding Information: We acknowledge the Centre for Advanced Light Microscopy at Ghent University (Belgium) for use of its microscopes and support for the microscopy experiments. The authors would like to acknowledge funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreements number (no.) 810685 (DelNam project) and no. 648124. S.S. acknowledges the support of a VLAIO grant (grant number: HBC.2017.0542). M.P. (FWO grant G036727N and FWO-SB grant 1S14318N), J.V.H. (FWO-SB grant 1S62519N), and J.C.F. (FWO grant 1210120N) thank the financial support by the Flemish Research Foundation. Conceptualization, L.R. K.R. and K.B.; investigation, L.R. M.P. A.H. G.G. and J.V.H.; project administration, L.R.; writing ? original draft, L.R.; writing ? review & editing, L.R. M.P. G.G. J.V.H. S.S. J.C.F. T.B. O.G.d.J. R.M.-B. E.M. P.V. S.C.D.S. B.V. K.R. and K.B.; funding acquisition, M.P. J.V.H. S.S. J.C.F. and K.B.; resources, G.G. O.G.d.J. R.M.-B. E.M. and P.V.; supervision, S.C.D.S. B.V. K.R. and K.B. The authors declare no competing interests. Publisher Copyright: © 2021 The Author(s) © 2021 The Author(s).
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